Presenilin associated membrane protein and uses thereof

ABSTRACT

Presenilin Associated Membrane Protein (PAMP), and nucleic acids encoding this protein, are provided. PAMP and PAMP nucleic acids provide diagnostic and therapeutic tools for evaluating and treating or preventing neurodegenerative diseases. In a specific embodiment, mutations in PAMP are diagnostic for Alzheimer&#39;s Disease or spina bifida. The invention further relates to screening, particularly using high-throughput screens and transgenic animal models, for compounds that modulate the activity of PAMP and presenilins. Such compounds, or gene therapy with PAMP, can be used in treating neurodegenerative diseases, particularly Alzheimer&#39;s Disease. In addition, the invention provides PAMP mutants, nucleic acids encoding for PAMP mutants, and transgenic animals expressing PAMP mutants, which in a preferred aspect result in biochemical changes similar to those induced by mutations in βAPP, PS1, or PS2, associated with familial Alzheimer&#39;s disease.

This patent application claims the priority of U.S. provisional patentapplication Nos. 60/127,452, filed Apr. 1, 1999 and No. 60/173,826,filed Dec. 30, 1999, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the field of neurological andphysiological dysfunctions associated with neuropsychiatric anddevelopmental diseases, especially Alzheimer's Disease. Moreparticularly, the invention is concerned with the identification ofproteins associated with neuropsychiatric and developmental diseases,especially Alzheimer's Disease, and relates to methods of diagnosingthese diseases, and to methods of screening for candidate compoundswhich modulate the interaction of a certain protein, specificallyPresenilin Associated Membrane Protein (“PAMP”), with presenilinproteins.

BACKGROUND OF THE INVENTION

Alzheimer's Disease (AD) is a degenerative disorder of the human centralnervous system characterized by progressive memory impairment andcognitive and intellectual decline during mid to late adult life(Katzman, N Eng J Med 1986;314:964-973). The disease is accompanied by aconstellation of neuropathologic features principal amongst which arethe presence of extracellular amyloid or senile plaques, andneurofibrillary tangles in neurons. The etiology of this disease iscomplex, although in some families it appears to be inherited as anautosomal dominant trait. Genetic studies have identified three genesassociated with the development of AD, namely: (1) β-amyloid precursorprotein (βAPP) (Chartier-Harlin et al., Nature 1991;353:844-846; Goateet al., Nature 1991;349:704-706; Murrell et al., Science 1991:254:97-99;Karlinsky et al., Neurology 1992;42:1445-1453; Mullan et al., NatureGenetics 1992;1:345-347); (2) presenilin-1 (PS1) (Sherrington et al.,Nature 1995;375:754-760); and (3) presenilin-2 (PS2) (Rogaev et al.,Nature 1995;376:775-778; Levy-Lehad et al., Science 1995; 269:970-973).

Abnormal processing of βAPP with overproduction of amyloid-β is also afeature of other CNS diseases, including inherited and sporadic forms ofamyloid angiopathy, which presents with intracerebral bleeding (Levy etal., Science 1990;248:1124-1126). Thus, abnormalities of presenilinproteins and PS-interacting proteins may affect these diseases as well.

The presenilin genes (PS1—PS1 and PS2—PS2) encode homologous polytopictransmembrane proteins that are expressed at low levels in intracellularmembranes including the nuclear envelope, the endoplasmic reticulum theGolgi apparatus and some as yet uncharacterized intracytoplasmicvesicles in many different cell types including neuronal andnon-neuronal cells (Sherrington et al., supra; Rogaev et al., supra,Levy-Lahad et al., supra; Doan et al., Neuron 1996;17:1023-1030; Walteret al., Molec. Medicine 1996;2:673-691; De Strooper et al., J. Biol.Chem. 1997;272:3590-3598; Lehmann et al., J.Biol.Chem.1997;272:12047-12051; Li et al., Cell 1997;90:917-927). Structuralstudies predict that the presenilins contain between six and eighttransmembrane (TM) domains organized such that the N-terminus, theC-terminus, and a large hydrophilic loop following the sixth TM domainare located in the cytoplasm or nucleoplasm, while the hydrophilic loopbetween TM1 and TM2 is located within the lumen of membranousintracellular organelles (Doan et al., 1996; De Strooper et al., 1997;et al., 1997).

Missense mutations in the PS1 and PS2 genes are associated with theinherited forms of early-onset AD (Sherrington et al., Nature1995;375:754-760; Rogaev, et al., Nature 1995;376:775-778; Levy-Lahad etal, Science 1995;269:970-973). Several lines of evidence have alsosuggested roles in developmental, apoptotic signalling and in theregulation of proteolytic cleavage of the β-amyloid precursor protein(βAPP) (Levitan et al., Nature 1995;377:351-354; Wong et al., Nature1997;387:288-292; Shen et al., Cell 1997;89:629-639; Wolozin et al.,Science 1996;274:1710-1713; De Strooper et al., Nature1998;391:387-390). Nevertheless, it remains unclear just how theseputative functions are mediated, or how they relate to the abnormalmetabolism of the βAPP associated with PS1 and PS2 mutations (Martin etal., NeuroReport 995;7:217-220; Scheuner et al., Nature Med.1996;2:864-870; Citron et al., Nature Med. 1997;3:67-72; Duff et al.,Nature 1996;383:710-713; Borchelt et al., Neuron 1996;17:1005-1013).

P1 and PS2 interact specifically with at least two members of thearmadillo family proteins; neuronal plakophilin-related armadilloprotein (Paffenholtz et al., Differentiation 1997; 61: 293-304;Paffenholtz et al., Exp Cell Res 1999; 250: 452-464; Zhou et al.,Neuroreport 1997; 8: 2085-2090) and β-catenin, that are expressed inboth embryonic and post-natal tissues. Moreover, the domains of PS1 andPS2 that interact with these proteins have been identified. Mutations inPS1 and PS2 affect the translocation of β-catenin into the nucleus ofboth native cells and cells transfected with a mutant PS gene. Theseinteractions and effects are described in detail in co-pending commonlyassigned U.S. application Ser. No. 09/227,725, filed Jan. 8, 1999, thedisclosure of which is incorporated herein by reference.

The identification and cloning of normal as well as mutant PS1 and PS2genes and gene products are described in detail in co-pending commonlyassigned U.S. application Ser. Nos. 08/431,048, filed Apr. 28, 1995;Ser. No. 08/496,841, filed Jun. 28, 1995; Ser. No. 08/509,359, filedJul. 31, 1995; and Ser. No. 08/592,541, filed Jan. 26, 1996, thedisclosures of which are incorporated herein by reference.

There is speculation that onset of AD may be associated with aberrantinteractions between mutant presenilin proteins and normal forms ofPS-interacting proteins, and these changes may increase or decreaseinteractions present with normal PS1, or cause interaction with amutation-specific PS-interacting protein. Such aberrant interactionsalso may result from normal presenilins binding to mutant forms of thePS-interacting proteins. Therefore, mutations in the PS-interactingproteins may also be implicated in the development of AD.

While the identification of normal and mutant forms of PS proteins hasgreatly facilitated development of diagnostics and therapeutics, a needexists for new methods and reagents to more accurately and effectivelydiagnose and treat AD. In addition, further insights into PS proteinsand their interaction with other components may lead to new diagnosticand treatment methods for other related CNS diseases.

SUMMARY OF THE INVENTION

Applicants have discovered that PS1 and PS2 interact specifically with atransmembrane protein, herein referred to as “Presenilin AssociatedMembrane Protein” or “PAMP”, which is expressed in multiple tissues(e.g., brain, kidney, lung, etc.). The product of this gene is thereforeimplicated in the biochemical pathways affected in Alzheimer's Disease,and may also have a role in other dementias, amyloid angiopathies, anddevelopmental disorders such as spina bifida. This gene, therefore,presents a new therapeutic target for the treatment of Alzheimer'sDisease and other neurologic diseases. In addition, PAMP nucleic acids,proteins and peptides, antibodies to PAMP, cells transformed with PAMPnucleic acids, and transgenic animals altered with PAMP nucleic acidsthat possess various utilities, as described herein for the diagnosis,therapy and continued investigation of Alzheimer's Disease and otherneurodegenerative disorders. Furthermore, mutant PAMP nucleic acids,proteins, or peptides, cells transfected with vectors comprising mutantPAMP nucleic acids, transgenic animals expressing mutant PAMP orpeptides thereof, and their use in studying Alzheimer's Disease andother neurodegenerative disorders, or developing improved diagnostic ortherapeutic methods for such disorders, are presented herein.

Thus, the invention provides isolated and purified presenilin associatedmembrane protein (PAMP), or a functional fragment thereof, as well asnucleic acids encoding a PAMP. Preferred nucleotide and amino acidsequences are provided herein. The invention further provides probes andprimers for PAMP genes. Preferred embodiments include sequences of atleast 10, 15 or 20 consecutive nucleotides selected from the disclosedsequences.

The invention also provides isolated and purified mutant PAMP, or afunctional fragment thereof, as well as nucleic acids encoding a mutantPAMP, and probes and primers for PAMP genes. Preferred nucleotide andamino acid sequences are provided herein.

Using the nucleic acid and amino acid sequences disclosed herein,methods for identifying allelic variants or heterospecfic homologues ofa human PAMP and gene are provided. The methods may be practiced usingnucleic acid hybridization or amplification techniques, immunochemicaltechniques, or any other technique known in the art. The allelicvariants may include other normal human alleles as well as mutantalleles of PAMP genes which may be causative of Alzheimer's Disease orother CNS diseases. The heterospecific homologues may be from othermammalian species, such as mice, rats, dogs, cats or non-human primates,or may be from invertebrate species, such as Drosophila melanogaster orCaenorhabditis elegans. Thus, it is another object of the invention toprovide nucleic acids that encode allelic or heterospecific variants ofthe disclosed sequences, as well as the allelic or heterospecificproteins encoded by them.

The invention also provides vectors, and particularly expression vectors(e.g., cos-Tet vector), which include any of the above-described nucleicacids. It is a further object of the invention to provide vectors inwhich normal or mutant PAMP nucleic acid sequences are operably joinedto exogenous regulatory regions to produce altered patterns ofexpression, or to exogenous coding regions to produce fusion proteins.Conversely, it is another object to provide nucleic acids in which PAMPregulatory regions are operably joined to exogenous coding regions,including standard marker genes, to produce constructs in which theregulation of PAMP genes may be studied and used in assays ortherapeutics.

The invention further provides host cells and transgenic animalstransformed with any of the above-described nucleic acids of theinvention. The host cells may be prokaryotic or eukaryotic cells and, inparticular, may be gametes, zygotes, fetal cells, or stem cells usefulin producing transgenic animal models. In one embodiment, the transgenicanimal contains a transgene encoding a normal or mutant PAMP, which isexpressed in neural cells such that expression can be detected, e.g., bydetecting PAMP, mRNA, or protein, and more preferably by detecting aneuroprotective or a neurodegenerative phenotype. For example, theanimal might manifest one or more symptoms of a neurodegenerativedisease. The animal may be a vertebrate or an invertebrate. In apreferred embodiment, the transgenic animal is a mouse, which encodes ahuman PAMP. The transgenic animal may further comprise a secondtransgene encoding a normal or mutant PS1, PS2, or βAPP.

In another embodiment, the invention provides an animal containing anucleic acid that expresses a PAMP or a mutant PAMP at a higher or lowerlevel relative to expression level in a wild-type animal. The animal maybe prepared by homologous recombination mediated targeting of endogenousPAMP nucleic acid. In a preferred embodiment, the animal is prepared bytranslocation of P-elements or chemical mutagenesis.

The invention also provides a reconstituted system for measuring PAMPactivity, comprising PAMP, a mutant PAMP, or functional fragmentsthereof, and a PAMP substrate. The reconstituted system may be a wholecell. Preferably, the whole cell contains a first nucleic acid thatexpresses said PAMP and a second nucleic acid that expresses thesubstrate. Preferably, the substrate comprises PS1 protein, PS2 protein,βAPP, or a surrogate synthetic substrate protein such as Notch, whichundergoes proteolytic processing events similar to those of βAPP (HaassC and Selkoe D J. Nature 1998; 391: 387-390; De Strooper B, et al.,Nature 1999; 398:518-522; Song W, et al., Proc Natl Acad Sci USA 1999;96: 6959-6963.; Struhl G and Greenwald I, Nature 1999; 398: 522-525; YeY, et al., Nature 1999; 398: 525-529.).

The invention provides, in addition, a complex between a PAMP, or amutant PAMP, and an agent which provides a detectable conformational orfunctional change in the PAMP upon interaction with a substance beinganalyzed for activity st a neurodegenerative disease. The complex mayfurther comprise PS1 protein, PS2 protein or βAPP.

The invention also provides a method for detecting a mutation in PAMPassociated with Alzheimer's or a related neurological disorder,comprising obtaining a nucleic acid sample from an individual diagnosedwith or suspected of having a neurodegenerative disorder, and sequencinga gene encoding PAMP from said sample.

The invention also invention provides a method for diagnosingindividuals predisposed to or having a neurodegenerative disorder,comprising obtaining a nucleic acid sample from an individual diagnosedwith or suspected of having a neurodegenerative disorder, and sequencinga gene encoding PAMP from said sample.

The invention also provides a method for diagnosing individualspredisposed to or having a neurodegenerative disorder, comprisingobtaining cells that contain nucleic acid encoding PAMP, and undernon-pathological conditions, transcribe the nucleic acid, and measuringa level of transcriptional activity of the nucleic acid.

The invention further provides a method for diagnosing individualspredisposed to or having a neurodegenerative disorder, comprisingobtaining cells from an individual that express nucleic acid encodingPAMP, or isolating PAMP from said individual, and measuring PAMPactivity, for example PAMP expression levels. In an alternativeembodiment, the activity or abundance of a PAMP substrate may bemeasured.

The invention also provides a method for identifying putative agentshaving anti-neurodegenerative activity, comprising administering one ormore putative agents to a transgenic animal and detecting a change inPAMP activity.

The invention also provides a method for identifying putative agentshaving anti-neurodegenerative activity, comprising adding one or moresaid agents to the reconstituted system described above, and detecting achange in PAMP activity.

The invention also provides a method for identifying putative agentshaving anti-neurodegenerative activity, comprising adding one or moresaid agents to the complex described above, and detecting aconformational change in PAMP.

The invention also provides a method for identifying proteins thatinteract with PAMP, comprising contacting said substance to thereconstituted system discussed above, and detecting a change in PAMPactivity.

Further the invention provides for a method for identifying substancesthat modulate PAMP activity, comprising contacting a sample containingone or more substances with PAMP, or a PAMP mutant, or functionalfragments thereof, and a PAMP substrate, measuring PAMP activity, anddetermining whether a change in PAMP activity occurs. In a preferredembodiment, the substance is a PAMP inhibitor. In another preferredembodiment, the substance stimulates PAMP activity.

These and other aspects of the invention are further elaborated in theDetailed Description of the Invention and Examples, infra.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Predicted amino acid sequences for human (SEQ IDNO:14), mouse (SEQ ID NO:16), D.melanogaster (SEQ ID NO:18) andC.elegans (SEQ ID NO:12) orthologues. Residues not conserved innon-human PAMP are blank, conserved residues are aligned, similarresidues are denoted by “+”.

DETAILED DESCRIPTION OF THE INVENTION

While PS1 and PS2 have been implicated in proper processing of βAPP, andmutations in these proteins have been associated with Alzheimer'sDisease, further understanding of the development and progression ofthis disease, as well as other neurodegenerative diseases, requires amore complete understanding of the functions of the presenilins andother proteins with which they interact. The present inventionadvantageously identifies such a protein.

PAMP

The invention is based, in part, on the discovery of a novel Type Itransmembrane protein that interacts with PS1 and PS2, and with the α-and β-secretase derived fragments of βAPP. The protein has been termed“Presenilin Associated Membrane Protein” (PAMP). As referred to herein,“PAMP” means a native or mutant full-length protein, or fragmentsthereof, that interacts with the PAMP-interacting domain of a presenilinprotein. PAMP is also known under the name “Nicastrin”. Human, murine,D. melanogaster and C. elegans orthologues are provided.

Experimental data indicates that PAMP, PS1, and PS2 exist in the samehigh molecular weight protein complex, and PAMP and PS1 are bothco-localized to intracellular membranes in the endoplasmic reticulum andGolgi apparatus. Abolition of functional expression of a C. eleganshomologue of this protein leads to the development of Notch-likedevelopmental defects. This shows that PAMP is also intimately involvedin the processing of not only βAPP, but also other molecules, such asNotch and its homologues. From expressed sequence tags (EST) databases,it is apparent that, like PS1 and PS2, PAMP is expressed in multipletissues.

Various structural features characterize PAMP (GenBank; Accession No.Q92542; SEQ ID NO: 14). The,nucleotide sequence (SEQ ID NO: 13) of humanPAMP predicts that the gene encodes a Type I transmembrane protein of709 amino acids (SEQ ID NO:14), the protein having a short hydrophilicC-terminus (˜20 residues), a hydrophobic transmembrane domain (15-20residues), and a longer N-terminal hydrophilic domain which containsseveral potentially functional sequence motifs as listed below inTable 1. The PAMP sequence also contains a Trp-Asp (WD) repeat (residue226), at least one “DTG” motif (residues 91-93) present in eukaryoticaspartyl proteases, as well as several “DTA/DTAE” motifs (residues480-482, 504-506) present in viral aspartyl proteases. There are alsofour conserved cysteine residues in the N-terminal hydrophilic domain(Cys195, Cys213, Cys230, and Cys248 in human PAMP) having a periodocityof 1617 residues, which may form a functional domain (e.g., a metalbinding domain or disulfide bridge for tertiary structurestabilization). Subdomains of PAMP have weak homologies to a variety ofpeptidases. For example, residues 322-343, 361-405, and 451-466 have 46%(p=0.03) similarity to another hypothetical protein; C. elegansaminopeptidase hydrolase precursor signal antigen transmembrane receptorzinc glycoprotein (SWISS-PROT; World Wide Web (www) expasy.ch/sprot;Accession No. Q93332).

TABLE 1 Potential functional sequence motifs in PAMP (SEQ ID NO:14).Potential function PAMP Residue N-asparaginyl glycosylation 44, 101,290, 492, 698, 964, 1353, 1772, 2209, 2675, 3183, 3715, 4279, 4854,5436, and 6050. Glycosaminoglycan attachment 403 Myristolation 4, 37,102, 226, 376, 548, 757, 1055, 1497, 1947, 2455, and 3035.Phosphorylation sites for 231 cAMP- and cGMP-dependent protein kinasePhosphorylation sites for 114, 383, 724, 1109, 1499, 1983, proteinkinase C 2598, and 3223 Phosphorylation sites for 7, 289, 652, 1026,1483, 1951, 2425, casein kinase II 3068, and 3717

The invention is further based on the, identification of conservedfunctional domains, based on comparison and evaluation of the predictedamino acid sequences of human (SEQ ID NO: 14), murine (SEQ ID NO: 16).D. melanogaster (SEQ ID NO: 18), and C. elegans (SEQ ID NO: 12)orthologues of PAMP. “PAMP” can be characterized by the presence ofconserved structural features, relative to orthologues from D.melanogaster and C. elegans. Nucleotide sequences encoding homologoushypothetical proteins exist in mice multiple EST, and C. elegans(GenBank; World Wide Web (www) ncbi.nlm.nih.gov; Accession No. Z75714;37% similarity, p=8.7e-²⁶) (Wilson et al., Nature 1994; 368: 32-38).These hypothetical murine and nematode proteins have a similar topologyand contain similar functional motifs to human PAMP. The existence ofsuch homology predicts that similar proteins will be detected in otherspecies including Xenopus, and Zebra fish, to mention a few suchpossibilities. By comparing the predicted amino acid sequences of human(SEQ ID NO: 14), murine (SEQ ID NO: 16), D. melanogaster (SEQ ID NO:18), and C. elegans (SEQ ID NO: 12) PAMP proteins, we have deduced aseries of conserved functional domains. One domain has chemicalsimilarities to cyclic nucleotide binding domains of other proteins, andmay have some regulatory role on a potential complex formed betweenPS1:PAMP and the C-terminal fragment of βAPP, derived either from a- orb-secretase. These putative functional domains are sites for therapeutictarget development by deploying drugs which might interact with thesesites to modulate βAPP processing via this complex.

The term “PAMP” also refers to functionally active fragments of theprotein. Such fragments include, but are not limited to, peptides thatcontain an epitope, e.g., as determined by conventional algorithms suchas hydrophilicity/hydrophobicity analysis for antibody epitopes, andamphipathicity or consensus algorithms for T cell epitopes (Spouge, etal., J. Immunol, 138:2204, 1987; Margalit, et al., J. Immunol.,138:2213, 1987; Rothbard, Ann. Inst. Pateur., 137E:518, 1986; Rothbardand Taylor, EMBO J., 7:93, 1988). More preferably, a functionally activefragment of PAMP is a conserved domain, relative to the D. melanogasterand C. elegans orthologues. A specific functionally active fragment ofPAMP is a fragment that interacts with PS1 or PS2, or both.

PAMP also encompasses naturally occurring variants, including othermammalian PAMPs (readily identified, as shown herein for murine PAMP,based on the presence of the structural features set forth above),allelic variants of PAMP from other human sources (including variantscontaining polymorphisms that are predictive of disease propensity or ofresponse to pharmacological agents), and mutant forms of PAMP or PAMPgenes that are associated with neurological diseases and disorders (suchas spina bifida), particularly neurodegenerative diseases (such as AD).Also included are artificial PAMP mutants created by standard techniquessuch as site directed mutagenesis or chemical synthesis.

A PAMP “substrate” may be a polypeptide or protein, or any other type ofcompound, with which PAMP interacts physiologically. Examples of PAMPsubstrates include PS1, PS2, and βAPP. Furthermore, A PAMP “ligand” maybe a polypeptide, protein, lipid, carbohydrate, vitamin, mineral, aminoacid, or any other type of compound which binds to PAMP Hypothetically,PAMP may function as a receptor which modulates PS1/PS2/βAPP processingin response to signal (ligand) dependent interactions with PAMP.

Definitions

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfilly in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1985)); Transcription And Translation (B. D. Hames & S. J. Higgins,eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

If appearing herein, the following terms shall have the definitions setout below.

The use of italics (e.g., PAMP) indicates a nucleic acid molecule (cDNA,mRNA, gene, etc.); normal text (e.g., PAMP) indicates the polypeptide orprotein.

In a specific embodiment, the term “about” or “approximately” meanswithin 20%, preferably within 10%, and more preferably within 5% of agiven value or range. Alternatively, particularly in biological systemswhich are often responsive to order of magnitude changes, the term aboutmeans within an order of magnitude of a given value, preferably within amultiple of about 5-fold, and more preferably within a factor of about2-fold of a given value.

As used herein, the term “isolated” means that the referenced materialis free of components found in the natural environment in which thematerial is normally found. In particular, isolated biological materialis free of cellular components. In the case of nucleic acid molecules,an isolated nucleic acid includes a PCR product, an isolated mRNA, acDNA, or a restriction fragment. In another embodiment, an isolatednucleic acid is preferably excised from the chromosome in which it maybe found, and more preferably is no longer joined to non-regulatory,non-coding regions, or to other genes, located upstream or downstream ofthe gene contained by the isolated nucleic acid molecule when found inthe chromosome. In yet another embodiment, the isolated nucleic acidlacks one or more introns. Isolated nucleic acid molecules can beinserted into plasmids, cosmids, artificial chromosomes, and the like.Thus, in a specific embodiment, a recombinant nucleic acid is anisolated nucleic acid. An isolated protein may be associated with otherproteins or nucleic acids, or both, with which it associates in thecell, or with cellular membranes if it is a membrane-associated protein.An isolated organelle, cell, or tissue is removed from the anatomicalsite in which it is found in an organism. An isolated material may be,but need not be, purified.

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate unrelated materials,i.e., contaminants. For example, a purified protein is preferablysubstantially free of other proteins or nucleic acids with which it isassociated in a cell; a purified nucleic acid molecule is preferablysubstantially free of proteins or other unrelated nucleic acid moleculeswith which it can be found within a cell.

As used herein, the term “substantially free” is used operationally, inthe context of analytical testing of the material. Preferably, purifiedmaterial substantially free of contaminants is at least 50% pure; morepreferably, at least 90% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, or used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells can further be used for screening or functional assays, asdescribed infra. A host cell has been “transfected” by exogenous orheterologous DNA when such DNA has been introduced inside the cell. Acell has been “transformed” by exongenous or heterologous DNA when thetransfected DNA is expressed and effects a function or phenotype on thecell in which it is expressed. The term “expression system” means a hostcell transformed by a compatible expression vector and cultured undersuitable conditions e.g. for the expression of a protein coded for byforeign DNA carried by the vector and introduced to the host cell.

Proteins and polypeptides can be made in the host cell by expression ofrecombinant DNA. As used herein, the term “polypeptide” refers to anamino acid-based polymer, which can be encoded by a nucleic acid orprepared synthetically. Polypeptides can be proteins, protein fragments,chimeric proteins, etc. Generally, the term “protein” refers to apolypeptide expressed endogenously in a cell, e.g., the naturallyoccurring form (or forms) of the amino acid-based polymer.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

The coding sequences herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the it naturally occurring nucleotides with an analog, andinternucleotide modifications.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of ribonucleicacids or amino acids which comprise all or part of one or more proteins,and may or may not include regulatory DNA sequences, such as promotersequences, which determine for example the conditions under which thegene is expressed.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background.

A coding sequence is “under the control” or “operatively associatedwith” of transcriptional and translational control sequences in a cellwhen RNA polymerase transcribes the coding sequence into mRNA, whichthen may be trans-RNA spliced (if it contains introns) and translatedinto the protein encoded by the coding sequence.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence toa host cell, so that the host cell will express the introduced gene orsequence to produce a desired substance, typically a protein or enzymecoded by the introduced gene or sequence. The introduced gene orsequence may also be called a “cloned”, “foreign”, or “heterologous”gene or sequence, and may include regulatory or control sequences usedby a cell's genetic machinery. The gene or sequence may includenonfunctional sequences or sequences with no known function. A host cellthat receives and expresses introduced DNA or RNA has been “transformed”and is a “transformant” or a “clone.” The DNA or RNA introduced to ahost cell can come from any source, including cells of the same genus orspecies as the host cell, or cells of a different genus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g., a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence. Vectors include plasmids, phages, viruses, etc. A “cassette”refers to a DNA coding sequence or segment of DNA that codes for anexpression product that can be inserted into a vector at definedrestriction sites. The cassette restriction sites are designed to ensureinsertion of the cassette in the proper reading frame. Generally,foreign DNA is inserted at one or more restriction sites of the vectorDNA, and then is carried by the vector into a host cell along with thetransmissible vector DNA. A segment or sequence of DNA having insertedor added DNA, such as an expression vector, can also be called a “DNAconstruct.” Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes.

A “knockout mammal” is a mammal (e.g., mouse) that contains within itsgenome a specific gene that has been inactivated by the method of genetargeting (see, e.g., U.S. Pat. No. 5,777,195 and U.S. Pat. No.5,616,491). A knockout mammal includes both a heterozygote knockout(i.e., one defective allele and one wild-type allele) and a homozygousmutant. Preparation of a knockout mammal requires first introducing anucleic acid construct that will be used to suppress expression of aparticular gene into an undifferentiated cell type termed an embryonicstem cell. This cell is then injected into a mammalian embryo. Amammalian embryo with an integrated cell is then implanted into a fostermother for the duration of gestation. Zhou, et al. (Genes andDevelopment, 9:2623-34, 1995) describes PPCA knock-out mice. Knockoutmice can be used to study defects in neurological development orneurodegenerative diseases. Disease phenotypes that develop can providea platform for further drug discovery.

The term “knockout” refers to partial or complete suppression of theexpression of at least a portion of a protein encoded by an endogenousDNA sequence in a cell. The term “knockout construct” refers to anucleic acid sequence that is designed to decrease or suppressexpression of a protein encoded by endogenous DNA sequences in a cell.The nucleic acid sequence used as the knockout construct is typicallycomprised of (1) DNA from some portion of the gene (exon sequence,intron sequence, and/or promoter sequence) to be suppressed and (2) amarker sequence used to detect the presence of the knockout construct inthe cell. The knockout construct is inserted into a cell, and integrateswith the genomic DNA of the cell in such a position so as to prevent orinterrupt transcription of the native DNA sequence. Such insertionusually occurs by homologous recombination (i.e., regions of theknockout construct that are homologous to endogenous DNA sequenceshybridize to each other when the knockout construct is inserted into thecell and recombine so that the knockout construct is incorporated intothe corresponding position of the endogenous DNA). The knockoutconstruct nucleic acid sequence may comprise 1) a full or partialsequence of one or more exons and/or introns of the gene to besuppressed, 2) a full or partial promoter sequence of the gene to besuppressed, or 3) combinations thereof. Typically, the knockoutconstruct is inserted into an embryonic stem cell (ES cell) and isintegrated into the ES cell genomic DNA, usually by the process ofhomologous recombination. This ES cell is then injected into, andintegrates with, the developing embryo.

Generally, for homologous recombination, the DNA will be at least about1 kilobase (kb) in length and preferably 3-4 kb in length, therebyproviding sufficient complementary sequence for recombination when theknockout construct is introduced into the genomic DNA of the ES cell.

A “knock-in” mammal is a mammal in which an endogenous gene issubstituted with a heterologous gene or a modified variant of theendogenous gene (Roemer et al., New Biol. 3:331, 1991). Preferably, theheterologous gene is “knocked-in” to a locus of interest, for exampleinto a gene that is the subject of evaluation of expression or function,thereby linking the heterologous gene expression to transcription fromthe appropriate promoter (in which case the gene may be a reporter gene;see Elefanty et al., Proc Natl Acad Sci USA 95:11897,1998). This can beachieved by homologous recombination, transposon (Westphal and Leder,Curr Biol 7:530, 1997), using mutant recombination sites (Araki et al.,Nucleic Acids Res 25:868, 1997) or PCR (Zhang and Henderson,Biotechniques 25:784, 1998).

The phrases “disruption of the gene” and “gene disruption” refer toinsertion of a nucleic acid sequence into one region of the native DNAsequence (usually one or more exons) and/or the promoter region of agene so as to decrease or prevent expression of that gene in the cell ascompared to the wild-type or naturally occurring sequence of the gene.By way of example, a nucleic acid construct can be prepared containing aDNA sequence encoding an antibiotic resistance gene which is insertedinto the DNA sequence that is complementary to the DNA sequence(promoter and/or coding region) to be disrupted. When this nucleic acidconstruct is then transfected into a cell, the construct will integrateinto the genomic DNA. Thus, some progeny of the cell will no longerexpress the gene, or will express it at a decreased level, as the DNA isnow disrupted by the antibiotic resistance gene.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is a such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature. In the context of the presentinvention, an gene is heterologous to the recombinant vector DNA inwhich it is inserted for cloning or expression, and it is heterologousto a host cell containing such a vector, in which it is expressed, e.g.,a CHO cell.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g. DNA, or any process, mechanism, or result of such achange. This includes gene mutations, in which the structure (e.g., DNAsequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g., protein) expressedby a modified gene or DNA sequence. The term “variant” may also be usedto indicate a modified or altered gene, DNA sequence, enzyme, cell,etc., i.e., any kind of mutant.

“Sequence-conservative variants” of a polynucleotide sequence are thosein which a change of one or more nucleotides in a given codon positionresults in no alteration in the amino acid encoded at that position.

“Function-conservative variants” are those in which a given amino acidresidue in a protein or enzyme has been changed without altering theoverall conformation and function of the polypeptide, including, but notlimited to, replacement of an amino acid with one having similarproperties (such as, for example, polarity, hydrogen bonding potential,acidic, basic, hydrophobic, aromatic, and the like). Amino acids withsimilar properties are well known in the art. For example, arginine,histidine and lysine are hydrophilic-basic amino acids and may beinterchangeable. Similarly, isoleucine, a hydrophobic amino acid, may bereplaced with leucine, methionine or valine. Such changes are expectedto have little or no effect on the apparent molecular weight orisoelectric point of the protein or polypeptide. Amino acids other thanthose indicated as conserved may differ in a protein or enzyme so thatthe percent protein or amino acid sequence similarity between any twoproteins of similar function may vary and may be, for example, from 70%to 99% as determined according to an alignment scheme such as by theCluster Method, wherein similarity is based on the MEGALIGN algorithm. A“function-conservative variant” also includes a polypeptide or enzymewhich has at least 60% amino acid identity as determined by BLAST(Altschul S F, et al., J Mol Biol 1990; 215: 403-410) or FASTAalgorithms, preferably at least 75%, most preferably at least 85%, andeven more preferably at least 90%, and which has the same orsubstantially similar properties or functions as the native or parentprotein or enzyme to which it is compared.

An “ortholog” to a protein means a corresponding protein from anotherspecies. Orthologous proteins typically have similar functions indifferent species, and can also be substantially homologous.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., Cell 50:667, 1987). Such proteins (and their encoding genes)have sequence homology, as reflected by their sequence similarity,whether in terms of percent similarity or the presence of specificresidues or motifs. Motif analysis can be performed using, for example,the program BLOCKS (www.blocks.fhcrc.org).

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., supra). However, in common usageand in the instant application, the term “homologous,” when modifiedwith an adverb such as “highly,” may refer to sequence similarity andmay or may not relate to a common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 80%, and mostpreferably at least about 90 or 95% of the nucleotides match over thedefined length of the DNA sequences, as determined by sequencecomparison algorithms, such as BLAST, FASTA, DNA Strider, etc. Sequencesthat are substantially homologous can be identified by comparing thesequences using standard software available in sequence data banks, orin a Southern hybridization experiment under, for example, stringentconditions as defined for that particular system.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than80% of the amino acids are identical, or greater than about 90% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program, ProteinPredict(dodo.cmpc.columbia.edu/predictprotein), or any of the programsdescribed above (BLAST, FASTA, etc.).

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m)(melting temperature) of 55° C., can be used. Moderate stringencyhybridization conditions correspond to a higher T_(m) and highstringency hybridization conditions correspond to the highest T_(m).Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of T_(m) for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherT_(m)) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA For hybrids of greater than 100 nucleotides inlength, equations for calculating T_(m) have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridization with shorter nucleic acids,i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). A minimum lengthfor a hybridizable nucleic acid is at least about 10 nucleotides;preferably at least about 15 nucleotides; and more preferably the lengthis at least about 20 nucleotides.

The present invention provides antisense nucleic acids (includingribozymes), which may be used to inhibit expression of PAMP, e.g., todisrupt a cellular process (such disruption can be used in an animalmodel or therapeutically). An “antisense nucleic acid” is a singlestranded nucleic acid molecule which, on hybridizing under cytoplasmicconditions with complementary bases in an RNA or DNA molecule, inhibitsthe latter's role. If the RNA is a messenger RNA transcript, theantisense nucleic acid is a countertranscript or mRNA-interferingcomplementary nucleic acid. As presently used, “antisense” broadlyincludes RNA-RNA interactions, RNA-DNA interactions, ribozymes andRNase-H mediated arrest. Antisense nucleic acid molecules can be encodedby a recombinant gene for expression in a cell (e.g., U.S. Pat. No.5,814,500; U.S. Pat. No. 5,811,234), or alternatively they can beprepared synthetically (e.g., U.S. Pat. No. 5,780,607).

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 10, preferably at least 15, and more preferably atleast 20 nucleotides, preferably no more than 100 nucleotides, that ishybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNAmolecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. In one embodiment, a labeled oligonucleotide can be used asa probe to detect the presence of a nucleic acid. In another embodiment,oligonucleotides (one or both of which may be labeled) can be used asPCR primers, e.g., for cloning full length or a fragment of a protein orpolypeptide. In a further embodiment, an oligonucleotide of theinvention can form a triple helix with a nucleic acid (genomic DNA ormRNA) encoding a protein or polypeptide. Generally, oligonucleotides areprepared synthetically, preferably on a nucleic acid synthesizer.Accordingly, oligonucleotides can be prepared with non-naturallyoccurring phosphoester analog bonds, such as thioester bonds, etc.Furthermore, the oligonucleotides herein may also be modified with alabel capable of providing a detectable signal, either directly orindirectly. Exemplary labels include radioisotopes, fluorescentmolecules, biotin, and the like.

Specific non-limiting examples of synthetic oligonucleotides envisionedfor this invention include oligonucleotides that containphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl, or cycloalkl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are those withCH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂, CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂and O—N(CH₃)—CH₂CH₂ backbones (where phosphodiester is O—PO₂—O—CH₂).U.S. Pat. No. 5,677,437 describes heteroaromatic olignucleosidelinkages. Nitrogen linkers or groups containing nitrogen can also beused to prepare oligonucleotide mimics (U.S. Pat. No. 5,792,844 and No.5,783,682). U.S. Pat. No. 5,637,684 describes phosphoramidate andphosphorothioamidate oligomeric compounds. Also envisioned areoligonucleotides having morpholino backbone structures (U.S. Pat. No.5,034,506). In other embodiments, such as the peptide-nucleic acid (PNA)backbone, the phosphodiester backbone of the oligonucleotide may bereplaced with a polyamide backbone, the bases being bound directly orindirectly to the aza nitrogen atoms of the polyamide backbone (Nielsenet al., Science 254:1497, 1991). Other synthetic oligonucleotides maycontain substituted sugar moieties comprising one of the following atthe 2′ position: OH, SH SCH₃, F, OCN, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃where n is from 1 to about 10, C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—; S—, or N-alkyl;O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substituted silyl; a fluorescein moiety; an RNA cleaving group; areporter group; an intercalator; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Oligonucleotides may alsohave sugar mimetics such as cyclobutyls or other carbocyclics in placeof the pentofuranosyl group. Nucleotide units having nucleosides otherthan adenosine, cytidine, guanosine, thymidine and uridine, such asinosine, may be used in an oligonucleotide molecule.

Presenilin Interacting Proteins

Mutant PS1 and PS2 genes, and their corresponding amino acid sequencesare described in Applicants' co-pending U.S. application Ser. No.08/888,077, filed Jul. 3, 1997, and incorporated herein by reference.Examples of PS1 mutations include I143T, M146L, L171P, F177S, A260V,C263R, P264L, P267S, E280A, E280G, A285V, L286V, Δ291-319, L322V, G384A,L392V, C410Y and 1439V. Examples of PS2 mutations include N141I, M239Vand I420T.

The methods of the present invention are not limited to mutantpresenilins wherein the PAMP-interacting domain is mutated relative tothe wild-type protein. For example, Applicants have observed thatmutations in PS1 (e.g., M146L) outside of the interacting domain (loop)also affect β-catenin translocation. These mutations probably disturbthe presenilin armadillo interactions by altering the function of a highMW complex which contains, e.g., the presenilin and armadillo proteins,as described in Yu et al., 1998, J. Biol. Chem. 273:16460-16475.Moreover, a comparison of the human PS1 (hPS1) and PS2 (hPS2) sequencesreveals that these pathogenic mutations are in regions of the PS1protein which are conserved in the PS2 protein. Therefore, correspondingmutations in corresponding regions of PS2 may also be expected to bepathogenic and are useful in the methods described herein.

Proteins that interact with the presenilins, i.e., PS-interactingproteins, include PAMP, the S5a subunit of the 26S proteasome (GenBank;Accession No. U51007), Rab11 (GenBank; Accession Nos. X56740 andX53143), retinoid X receptor B, also known as nuclear receptorco-regulator or MHC (GenBank Accession Nos. M84820, and X63522), GT24(GenBank Accession No. U.S.81004), β-catenin (Zhou et al., 1997, Neuro.Report (Fast Track) 8:1 025-1029 and Yu et al., supra) as well asarmadillo proteins. These and other PS1 binding proteins are describedin Applicants' copending commonly assigned U.S. application Ser. No.08/888,077, filed Jul. 3, 1997, as well as U.S. application Ser. No.08/592,541, filed Jan. 26, 1996, the disclosures of which areincorporated herein by reference.

PAMP Mutants

PAMP mutants may cause biochemical changes similar to those affectingthe onset or progression of Alzheimer Disease. Therefore, artificialPAMP mutations can potentially be used to generate cellular and othermodel systems to design treatments and preventions for Alzheimer Diseaserelated disorders. Such mutations may also be used for evaluatingwhether PAMP is involved in the pathogenesis of AD. Since the amyloid-β(Aβ) inducing mutations are found in amino acid residues of a soluble(non-membrane spanning) domain of PAMP, analysis of the normal structureof this domain and the effects of these and other nearby mutations onthe structure of this domain (and the other domains of PAMP) provideinformation for the design of specific molecular therapeutics.

In general, modifications of the sequences encoding the polypeptidesdescribed herein may be readily accomplished by standard techniques suchas chemical syntheses and site-directed mutagenesis. See Gillman et al.,1979, Gene 8:81-97; Roberts et al., 1987, Nature 328:731-734; and Innis(ed.), 1990, PCR Protocols: A Guide to Methods and Applications,Academic Press, New York. Most modifications are evaluated by routinescreening via an assay designed to select for the desired property.

Antibodies to PAMP

According to the invention, PAMP polypeptides produced recombinantly orby chemical synthesis, and fragments or other derivatives or analogsthereof including fission proteins and PAMP mutants, may be used as animmunogen to generate antibodies that recognize the PAMP polypeptide.Such antibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments, and an Fab expression library.Such an antibody is preferably specific for human PAMP, PAMP originatingfrom other species, or for post-translationally modified (e.g.phosphorylated, glycosylated) PAMP.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to PAMP polypeptide or derivative or analogthereof. For the production of antibody, various host animals can beimmunized by injection with the PAMP polypeptide, or a derivative (e.g.,fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the PAMPpolypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Antiseramay be collected at a chosen time point after immunization, and purifiedas desired.

For preparation of monoclonal antibodies directed toward the PAMPpolypeptide, or fragment, analog, or derivative thereof, any techniquethat provides for the production of antibody molecules by continuouscell lines in culture may be used. These include but are not limited tothe hybridoma technique originally developed by Kohler and Milstein(Nature 256:495-497, 1975), as well as the trioma technique, the humanB-cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983;Cote et al., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030, 1983), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96, 1985). Production of human antibodies by CDR grafting isdescribed in U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762 to Queenet al., and also in U.S. Pat. No. 5,225,539 to Winter and InternationalPatent Application PCT/WO91/09967 by Adau et al. In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals (International Patent Publication No. WO 89/12690,published 28 Dec. 1989). In fact, according to the invention, techniquesdeveloped for the production of “chimeric antibodies” (Morrison et al.,J. Bacteriol. 159:870, 1984); Neuberger et al., Nature 312:604-608,1984; Takeda et al., Nature 314:452-454, 1985) by splicing the genesfrom a mouse antibody molecule specific for an PAMP polypeptide togetherwith genes from a human antibody molecule of appropriate biologicalactivity can be used; such antibodies are within the scope of thisinvention. Such human or humanized chimeric antibodies are preferred foruse in therapy of human diseases or disorders (described infra), sincethe human or humanized antibodies are much less likely than xenogenicantibodies to induce an immune response, in particular an allergicresponse, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No. 4,946,778) can be adapted to produce PAMPpolypeptide-specific single chain antibodies. An additional embodimentof the invention utilizes the techniques described for the constructionof Fab expression libraries (Huse et al., Science 246:1275-1281, 1989)to allow rapid and easy identification of monoclonal Fab fragments withthe desired specificity for an PAMP polypeptide, or its derivatives, oranalogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of an PAMP polypeptide, one may assay generatedhybridomas for a product which binds to an PAMP polypeptide fragmentcontaining such epitope. For selection of an antibody specific to anPAMP polypeptide from a particular species of animal, one can select onthe basis of positive binding with PAMP polypeptide expressed by orisolated from cells of that species of animal.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the PAMP polypeptide, e.g.,for Western blotting, imaging PAMP polypeptide in situ, measuring levelsthereof in appropriate physiological samples, etc. using any of thedetection techniques mentioned above or known in the art. Suchantibodies can be used to identify proteins that interact with PAMP, andto detect conformational or structural changes in PAMP.

In a specific embodiment, antibodies that agonize or antagonize theactivity of PAMP polypeptide can be generated. They can also be used toregulate or inhibit PAMP activity intracellular, i.e., the inventioncontemplates an intracellular antibody (intrabody), e.g., single chainFv antibodies(see generally, Chen, Mol. Med. Today, 3:160-167, 1997;Spitz et al., Anticancer Res., 16:3415-3422, 1996; Indolfi et al., Nat.Med., 2:634-635, 1996; Kijima et al., Pharmacol. Ther., 68:247-267,1995).

PAMP Diagnostic Assays

The nucleotide sequence and the protein sequence and the putativebiological activity of PAMP or PAMP mutants can all be used for thepurposes of diagnosis of individuals who are at-risk for, or whoactually have, a variety of neurodegenerative diseases (includingAlzheimer's disease, Lewy body variant, Parkinson's disease-dementiacomplex, amyotrophic lateral sclerosis etc.), neuropsychiatric diseases(schizophrenia, depression, mild cognitive impairment, benign senescentforgetfulness, age-associated memory loss, etc.), developmentaldisorders associated with defects in intracellular signal transductionmediated by factors such as Notch, Delta, Wingless, etc., and neoplasms(e.g., bowel cancer, etc.) associated with abnormalities of PS1/PAMP/PS2mediated regulation of cell death pathways. These diagnostic entitiescan be used by searching for alterations in: the nucleotide sequence ofPAMP; in the transcriptional activity of PAMP; in the protein level asmonitored by immunological means (e.g., ELISA and Western blots); in theamino acid sequence (as ascertained by Western blotting, amino acidsequence analysis, mass spectroscopy); and/or in the biological activityof the PAMP protein as measured by either in viva methods (e.g.,monitoring βAPP processing and the production of amyloid-β peptide (Aβ),or other suitable protein substrates for PAMP including Notch, etc.), orby in vitro assays (using either whole cell or cell-free assays tomeasure processing of suitable substrates including βAPP or partsthereof, and other proteins such as Notch). Any of these assays can alsobe performed in a transgenic animal model as well, e.g., to measure theeffect of a drug or a mutation or overexpression of a different gene invivo.

PAMP Screening Assays

Identification and isolation of PAMP provides for development ofscreening assays, particularly for high throughput screening ofmolecules that up- or down-regulate the activity of PAMP, e.g., bypermitting expression of PAMP in quantities greater than can be isolatedfrom natural sources, or in indicator cells that are speciallyengineered to indicate the activity of PAMP expressed after transfectionor transformation of the cells. Any screening technique known in the artcan be used to screen for PAMP agonists or antagonists. The presentinvention contemplates screens for small molecule ligands or ligandanalogs and mimics, as well as screens for natural ligands that bind toand agonize or antagonize the activity of PAMP in vivo. For example,natural products libraries can be screened using assays of the inventionfor molecules that agonize or antagonize PAMP activity.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” (Scott and Smith, Science249:386-390, 1990; Cwirla, et al., Proc. Natl. Acad. Sci., 87:6378-6382,1990; Devlin et al., Science, 49:404-406, 1990), very large librariescan be constructed (10⁶-10⁸ chemical entities). A second approach usesprimarily chemical methods, of which the Geysen method (Geysen et al.,Molecular Immunology 23:709-715, 1986; Geysen et al. J. ImmunologicMethod 102:259-274, 1987; and the method of Fodor et al. (Science251:767-773, 1991) are examples. Furka et al. (14th Int. Congress ofBiochemistry, Volume 5, Abstract FR:013, 1988; Furka, Int J. PeptideProtein Res. 37:487-493, 1991), Houghton (U.S. Pat. No. 4,631,211,issued December 1986) and Rutter et al. (U.S. Pat. No. 5,010,175, issuedApr. 23, 1991) describe methods to produce a mixture of peptides thatcan be tested as agonists or antagonists.

In another aspect, synthetic libraries (Needels et al., Proc. Natl.Acad. Sci. USA 90:10700-4, 1993; Ohlmeyer et al., Proc. Natl. Acad. Sci.USA 90:10922-10926, 1993; Lam et al., International Patent Publ. No. WO92/00252; Kocis et al., International Patent Publ. No. WO 9428028) andthe like can be used to screen for PAMP ligands according to the presentinvention.

Knowledge of the primary sequence of the protein, and the similarity ofthat sequence with proteins of known function, can provide an initialclue as to the inhibitors or antagonists of the protein. As noted above,identification and screening of antagonists is further facilitated bydetermining structural features of the protein, e.g., using X-raycrystallography, neutron diffraction, nuclear magnetic resonancespectrometry, and other techniques for structure determination. Thesetechniques provide for the rational design or identification of agonistsand antagonists.

The PAMP protein sequence (including parts thereof) can be used todeduce the structural organization and topology of PAMP through the useof a variety of techniques including CD spectroscopy, nuclear magneticresonance (NMR) spectroscopy, X-ray crystallography, and molecularmodeling. Sequences for PAMP or PAMP mutants can also be used toidentify proteins which interact with PAMP either in concert with PS1and PS2, or independently, using a variety of methods includingco-immunoprecipitation, yeast two hybrid interaction trap assays, yeastthree hybrid interaction trap assays, chemical cross-linking andco-precipitation studies, etc. These and other methods are describedmore fully in co-pending and commonly assigned U.S. application Ser. No.08/888,077, filed Jul. 3, 1997, and 09/227,725, filed Jan. 8, 1999, bothof which are incorporated herein by reference. Identification of theseinteracting partners will then lead to their use to further delineatethe biochemical pathways leading to the above-mentioned diseases.

Finally, the structural analysis of PAMP, when combined with structuralanalysis of PS1 and PS2, and other proteins which interact with PAMP orPAMP mutants, will identify the structural domains that mediateinteractions between these molecules and which also confer biologicalactivity on PAMP (or PAMP and these other molecules). These structuraldomains, and other functional domains, which can modulate the activityof these structural domains, can all be modified through a variety ofmeans, including but not limited to site-directed mutagenesis, in orderto either augment or reduce the biological activity. The structure andtopology of these domains can all be used as a basis for the rationaldesign of pharmaceuticals (small molecule conventional drugs or novelcarbohydrate, lipid, DNA/RNA or protein-based high molecular weightbiological compounds) to modulate (increase or decrease) the activity ofPAMP and/or the PAMP PS1/PS2 complex, and/or the activity of thePAMP/other protein complexes. For example, using structural predictioncalculations, possibly in conjunction with spectroscopic data likenuclear magnetic resonance, circular dichroism, and otherphysical-chemical structural data, or crystallographic data, or both,one can generate molecular models for the structure of PAMP. Thesemodels, in turn, are important for rational drug design. Drug candidatesgenerated using a rational drug design program can then be applied forthe treatment and/or prevention of the above-mentioned diseases, and canbe administered through a variety of means including: as conventionalsmall molecules through enteral or parenteral routes; via inclusion inliposome vehicles; through infusion in pumps inserted into variousorgans (e.g., via Omaya pumps inserted into the cerebral ventricles);via the transplantation of genetically-modified cells expressingrecombinant genes; or via the use of biological vectors (e.g.,retrovirus, adenovirus, adeno-associated virus, Lentivirus, or herpessimplex virus-based vectors) which allow targeted expression ofappropriately modified gene products in selected cell types. It shouldbe noted that the recombinant proteins described above may be thewild-type PAMP, a genetically-modified PAMP, a wild-type PS1/PS2, agenetically-modified PS1/PS2, or a specially-designed protein or peptidewhich is designed to interact with either the functional domains of PAMP(or the PAMP/PS1/PS2/other protein complex) or to interact with thedomains which modulate the activity of the functional domains of PAMP.

PAMP In Vitro and In Vivo Models

The PAMP nucleotide sequence can be used to make cell-free systems,transfected cell lines, and animal models (invertebrate or vertebrate)of the neurodegenerative and other diseases outlined above. These animaland cell models may involve over-expression of all or part of PAMP orPAMP mutants, e.g., as mini-gene cDNA transgene constructs under theregulation of suitable promoter elements carried in vectors such ascos-Tet for transgenic mice and pcDNA (Invitrogen, California) intransfected cell lines. Animal and cellular models can also be generatedby via homologous recombination mediated targeting of the endogenousgene to create artificially mutant sequences (knock-in gene targeting);or loss of function mutations (knock-out gene targeting); bytranslocation of P-elements; and by chemical mutagenesis. Animal,cellular and cell-free model systems can be used for a variety ofpurposes including the discovery of diagnostics and therapeutics forthis disease.

Included within the scope of this invention is a mammal in which two ormore genes have been knocked out or knocked in, or both. Such mammalscan be generated by repeating the procedures set forth herein forgenerating each knockout construct, or by breeding to mammals, each witha single gene knocked out, to each other, and screening for those withthe double knockout genotype.

Regulated knockout animals can be prepared using various systems, suchas the tet-repressor system (see U.S. Pat. No. 5,654,168) or the Cre-Loxsystem (see U.S. Pat. No. 4,959,317 and U.S. Pat. No. 5,801,030).

Transgenic mammals can be prepared for evaluating the molecularmechanisms of PAMP, and particularly human PAMP/PS1 or PAMP/PS2function. Such mammals provide excellent models for screening or testingdrug candidates. It is possible to evaluate compounds or diseases on“knockout” animals, e.g., to identify a compound that can compensate fora defect in PAMP activity. Alternatively, PAMP (or mutant PAMP), aloneor in combination with βAPP, PS1, and/or PS2, (double or tripletransgenics) “knock-in” mammals can be prepared for evaluating themolecular biology of this system in greater detail than is possible withhuman subjects. Both technologies permit manipulation of single units ofgenetic information in their natural position in a cell genome and toexamine the results of that manipulation in the background of aterminally differentiated organism. These animals can be evaluated forlevels of mRNA or protein expression, processing of βAPP, or developmentof a condition indicative of inappropriate gene expression, e.g., Notchphenotype or another phenotype as set forth above, or neurodegeneration,including cognitive deficits, learning or memory deficits, orneuromuscular deficits.

Various transgenic animal systems have been developed. Mice, rats,hamsters, and other rodents are popular, particularly for drug testing,because large numbers of transgenic animals can be bred economically andrapidly. Larger animals, including sheep, goats, pigs, and cows, havebeen made transgenic as well. Transgenic D.melanogaster and C.eleganscan also be made and, using known genetic methods, may offer the abilityto identify upstream and downstream modifiers of a PAMP phenotype.Transgenic animals can also be prepared by introducing the transgene ona vector; such animals, which are not modified in the germ line and areonly transiently transgenic, naturally cannot pass along the geneticinformation to their progeny.

In another series of embodiments, transgenic animals are created inwhich (i) a human PAMP, or a mutant human PAMP, is stably inserted intothe genome of the transgenic animal; and/or (ii) the endogenous PAMPgenes are inactivated and replaced with their human counterparts. See,e.g., Coffman, Semin. Nephrol. 17:404, 1997; Esther et al., Lab. Invest.74:953, 1996; Murakami et al., Blood Press. Suppl. 2:36, 1996. Suchanimals can be treated with candidate compounds and monitored for theeffects of such drugs on PAMP cavity.

PAMP Gene Therapy

As discussed above, abnormalities in PAMP expression and/or interactionswith PS1/PS2/βAPP are associated with severe neurological deficits.Thus, the present invention provides for treatment of such deficitseither with a drug discovered using a screening assay or transgenicanimal model, or both, as set forth above, or by replacing a defectivePAMP gene with a functional gene by gene therapy.

A gene encoding PAMP, a PAMP mutant, or alternatively a negativeregulator of PAMP such as an antisense nucleic acid, intracellularantibody (intrabody), or dominant negative PAMP (which may betruncated), can be introduced in vivo, ex vivo, or in vitro using aviral or a non-viral vector, e.g., as discussed above. Expression intargeted tissues can be effected by targeting the transgenic vector tospecific cells, such as with a viral vector or a receptor ligand, or byusing a tissue-specific promoter, or both. Targeted gene delivery isdescribed in International Patent Publication WO 95/28494, publishedOctober 1995.

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector, e.g.,adenovirus vector, to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin 12 (IL-12), interferon-γ (IFNγ), or anti-CD4 antibody, canbe administered to block humoral or cellular immune responses to theviral vectors (see, e.g., Wilson, Nature Medicine, 1995). In thatregard, it is advantageous to employ a viral vector that is engineeredto express a minimal number of antigens.

Herpes virus vectors. Because herpes virus is trophic for cells of thenervous system (neural cells), it is an attractive vector for deliveryof function PAMP genes. Various defective (non-replicating, and thusnon-infectious) herpes virus vectors have been described, such as adefective herpes virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell.Neurosci. 2:320-330, 1991; International Patent Publication No. WO94/21807, published Sep. 29, 1994; International Patent Publication No.WO 92/05263, published Apr. 2, 1994).

Adenovirus vectors. Adenoviruses are eukaryotic DNA viruses that can bemodified to efficiently deliver a nucleic acid of the invention to avariety of cell types in vivo, and has been used extensively in genetherapy protocols, including for targeting genes to neural cells.Various serotypes of adenovirus exist. Of these serotypes, preference isgiven to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) oradenoviruses of animal origin (see WO94/26914). Those adenoviruses ofanimal origin which can be used within the scope of the presentinvention include adenoviruses of canine, bovine, murine (example: Mavl,Beard et al., Virology 75 (1990) 81), ovine, porcine, avian, and simian(example: SAV) origin. Preferably, the adenovirus of animal origin is acanine adenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan orA26/61 strain (ATCC VR-800), for example). Various replication defectiveadenovirus and minimum adenovirus vectors have been described for genetherapy (WO94/26914, WO95/02697, WO94/28938, WO94/28152, WO94/12649,WO95/02697 WO96/22378). The replication defective recombinantadenoviruses according to the invention can be prepared by any techniqueknown to the person skilled in the art (Levrero et al., Gene 101:1951991; EP 185 573; Graham, EMBO J. 3:2917, 1984; Graham et al., J. Gen.Virol. 36:59 1977). Recombinant adenoviruses are recovered and purifiedusing standard molecular biological techniques, which are well known toone of ordinary skill in the art.

Adeno-associated viruses. The adeno-associated viruses (AAV) are DNAviruses of relatively small size which can integrate, in a stable andsite-specific manner, into the genome of the cells which they infect.They are able to infect a wide spectrum of cells without inducing anyeffects on cellular growth, morphology or differentiation, and they donot appear to be involved in human pathologies. The AAV genome has beencloned, sequenced and characterized. The use of vectors derived from theAAVs for transferring genes in vitro and in vivo has been described (seeWO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No.5,139,941, EP 488 528). The replication defective recombinant AAVsaccording to the invention can be prepared by co-transfecting a plasmidcontaining the nucleic acid sequence of interest flanked by two AAVinverted terminal repeat (ITR) regions, and a plasmid carrying the AAVencapsidation genes (rep and cap genes), into a cell line which isinfected with a human helper virus (for example an adenovirus). The AAVrecombinants which are produced are then purified by standardtechniques.

Retrovirus vectors. In another embodiment the gene can be introduced ina retroviral vector, e.g., as described in Anderson et al., U.S. Pat.No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin et al., U.S. Pat.No. 4,650,764, Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al.,1988, J. Virol. 62:1120; Temin et al., U.S. Pat. No. 5,124,263, EP453242, EP178220; Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick,BioTechnology 3 (1985) 689; International Patent Publication No. WO95/07358, published Mar. 16, 1995, by Dougherty et al., and Kuo et al.,1993, Blood 82:845. The retroviruses are integrating viruses whichinfect dividing cells. The retrovirus genome includes two LTRs, anencapsidation sequence and three coding regions (gag, pol and env). Inrecombinant retroviral vectors, the gag, pol and env genes are generallydeleted, in whole or in part, and replaced with a heterologous nucleicacid sequence of interest. These vectors can be constructed fromdifferent types of retrovirus, such as MoMuLV (“murine Moloney leukemiavirus”), MEV (“murine Moloney sarcoma virus”), HaSV (“Harvey sarcomavirus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcoma virus”) andFriend virus. Suitable packaging cell lines have been described in theprior art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719);the PsiCRIP cell line (WO 90/02806) and the GP+envAm-12 cell line (WO89/07150). In addition, the recombinant retroviral vectors can containmodifications within the LTRs for suppressing transcriptional activityas well as extensive encapsidation sequences which may include a part ofthe gag gene (Bender et al., J. Virol. 61:1639, 1987). Recombinantretroviral vectors are purified by standard techniques known to thosehaving ordinary skill in the art.

Retrovirus vectors can also be introduced by recombinant DNA viruses,which permits one cycle of retroviral replication and amplifiestransfection efficiency (see WO 95/22617, WO 95/26411, WO 96/39036, WO97/19182).

Lentivirus vectors. In another embodiment, lentiviral vectors are can beused as agents for the direct delivery and sustained expression of atransgene in several tissue types, including brain, retina, muscle,liver and blood. The vectors can efficiently transduce dividing andnon-dividing cells in these tissues, and maintain long-term expressionof the gene of interest. For a review, see, Naldini, Curr. Opin.Biotechnol., 9:457-63, 1998; see also Zufferey, et al., J. Virol.,72:9873-80, 1998). Lentiviral packaging cell lines are available andknown generally in the art. They facilitate the production of high-titerlentivirus vectors for gene therapy. An example is atetracycline-inducible VSV-G pseudotyped lentivirus packaging cell linewhich can generate virus particles at titers greater than 106 IU/ml forat least 3 to 4 days (Kafri, et al., J. Virol., 73: 576-584, 1999). Thevector produced by the inducible cell line can be concentrated as neededfor efficiently transducing nondividing cells in vitro and in vivo.

Non-viral vectors. A vector can be introduced in vivo in a non-viralvector, e.g., by lipofection, with other transfection facilitatingagents (peptides, polymers, etc.), or as naked DNA Synthetic cationiclipids can be used to prepare liposomes for in vivo transfection, withtargeting in some instances (Felgner, et. al., Proc. Natl. Acad. Sci.U.S.A. 84:7413-7417, 1987; Felgner and Ringold, Science 337:387-388,1989; see Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031,1988; Ulmer et al., Science 259:1745-1748, 1993). Useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO95/18863 and WO96/17823, and in U.S.Pat. No. 5,459,127. Other molecules are also useful for facilitatingtransfection of a nucleic acid in vivo, such as a cationic oligopeptide(e.g. International Patent Publication WO95/21931), peptides derivedfrom DNA binding proteins (e.g., International Patent PublicationWO96/25508), or a cationic polymer (e.g., International PatentPublication WO95/21931). Recently, a relatively low voltage, highefficiency in vivo DNA transfer technique, termed electrotransfer, hasbeen described (Mir et al., C.P. Acad. Sci., 321:893, 1998; WO 99/01157;WO 99/01158; WO 99/01175). DNA vectors for gene therapy can beintroduced into the desired host cells by methods known in the art,e.g., electroporation, microinjection, cell fusion, DEAE dextran,calcium phosphate precipitation, use of a gene gun (ballistictransfection), or use of a DNA vector transporter (see, e.g., Wu et al.,J. Biol. Chem. 267:963-967, 1992; Wu and Wu, J. Biol. Chem.263:14621-14624, 1988; Hartmut et al., Canadian Patent Application No.2,012,311, filed Mar. 15, 1990; Williams et al., Proc. Natl. Acad. Sci.USA 88:2726-2730, 1991). Receptor-mediated DNA delivery approaches canalso be used (Curiel et al., Hum. Gene Ther. 3:147-154, 1992; Wu and Wu,J. Biol. Chem. 262:4429-4432, 1987). U.S. Pat. Nos. 5,580,859 and5,589,466 disclose delivery of exogenous DNA sequences, free oftransfection facilitating agents, in a mammal.

EXAMPLES

The present invention will be further understood by reference to thefollowing examples, which are provided as exemplary of the invention andnot by way of limitation.

Example 1 A Novel PAMP that Mediates βAPP Processing and Notch/Clp1Signal Transduction

This example shows that both PS1 and PS2 interact with a novel Type Itransmembrane protein, PAMP, and that this novel protein also interactswith α- and β-secretase derived fragments of βAPP. We also show thatabolition of functional expression of the C. elegans homologue of theprotein leads to a developmental phenotype (anterior pharynx 2-aph2)which is thought to be due to inhibition of the glp/Notch signalingpathway. This novel protein is therefore positioned to mediate both thegain of function and loss of function phenotypes associated withpresenilin missense mutations and presenilin knockouts, respectively.

Materials and Methods

Antibodies against PS1, PS2 and βAPP. An antibody, termed 1142, directedagainst PS1, was raised to a peptide segment corresponding to residues30-45 of PS1 (Levesque et al., J Neurochem 1998: 72:999-1008; Yu et al.,Biol Chem1998; 273:16470-16475). The peptide was synthesized bysolid-phase techniques and purified by reverse phase high pressureliquid chromatography (HPLC). Peptide antigens were linked to keyholelimpet hemocyanin (KLH) and used, in combination with complete Freud'sadjuvant, to innoculate New Zealand White rabbits. Antisera from threerabbits was pooled, ammonium precipitated and the antibody was purifiedwith Sulfo-link (Pierce) agarose-peptide affinity columns. Otherantibodies used include antibody 369, a polyclonal rabbit-anti-humanantibody directed against the C-terminus of human βAPP (Buxbaum et al.,Proc. Natl. Acad. Sci. USA 1990; 87: 6003-6007); antibody 14 (Ab14), arabbit polyclonal antibody raised against residues 1-25 of human PS1(Seeger et al., Proc. Natl. Acad. Sci. 1997, 94: 5090-5094); antibodyα-PS1-CTF, a polyclonal rabbit antibody directed against the PS1 loop;and antibody DT2, a monoclonal antibody raised to a GST-fusion proteincontaining the PS2 N-terminal sequence from residues 1-87.

Preparation of presenilin associated components. To identify membraneassociated components of the presenilin complex, an immunoaffinityprocedure was used to extract PS1 and tightly associated membraneproteins from semi-purified intracellular membrane fractions. Humanembryonic kidney cells (HEK) 293 (ATCC) with a stable over-expression ofmoderate level wild type human PS1, were grown to confluence, washedtwice with ice-cold phosphate-buffered saline, and then homogenized withBuffer A (0.25 M sucrose, 20 mM HEPES pH 7.2, 2 mM EGTA, 2 mM EDTA, 1 mMDTT, and a protease inhibitor cocktail containing 5 μml each ofLeupeptin, Antipain, pepstatin A, Chymostin, E64, Aprotinin, and 60μg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF)). The cellhomogenates were centrifuged 1000×g for 10 minutes to remove celldebris. The supernatant was then centrifuged 10,000×g for 60 minutes.The resulting membrane pellet was resuspended in Buffer B (20 mM HEPESpH 7.2, 1 M KCl, 2 mM EGTA, 2 mM EDTA, 1 mM DTT, and protease inhibitorcocktail as above) and incubated for 45 minutes with agitation at 4° C.Cell membranes were collected again by centrifugation at 107,000×g for60 minutes. The cell membranes were then lysed on ice for 60 minuteswith Buffer C (1% Digitonin, 20 mM HEPES pH 7.2, 100 mM KCl, 2 mM EGTA,2 mM EDTA, 1 mM DTT, and protease inhibitors cocktail). After spinning10,000×g for 15 minutes, the protein extract was adjusted with Buffer Cto contain 5 mg/ml protein. A total of 0.5 g of protein was obtained.

Isolation. The extracted proteins were then subjected to fractionationwith 10-40% glycerol gradient containing 0.5% Digitonin as described (YuG, et al., J Biol Chem 1998; 273: 16470-16475). After being verified byWestern blotting with anti-PS1 antibodies, the peak fractions containingPS1 were pooled and incubated overnight with Protein A/G agarose coupledwith either antibody 1142 or a control IgG purified from preimmunerabbit serum. The Protein A/G agarose beads were washed three times withBuffer D (1% Digitonin, 20 mM HEPES pH 7.2, 100 mM KCl, proteaseinhibitors cocktail), and three times with Buffer E (0.5% Digitonin,0.5% CHAPS, 20 mM HEPES pH 7.2, 100 mM KCl, 10 mM CaCl₂, 5 mM MgCl₂, andthe protease inhibitor cocktail as above). Isolated protein complexeswere eluted from the beads with 0.1M Glycine-HCl, pH 3.0, and thenneutralized with 1M Tris. Proteins were then separated by Tris-GlycineSDS-PAGE gels and stained with silver stain and Coomassie Blue stain.The staining of the immuno-purified proteins displayed two intense bandsin addition to those of the presenilin holoprotein and its fragments.

Sequence analysis. Individual protein bands were cut out and analyzedwith solid-phase extraction capillary electrophoresis massspectrometry/mass spectrometry (SPE-CE-MS/MS). Briefly, protein bandswere first digested in-gel with trypsin; the digested proteins wereextracted and dried in a speed vacuum down to concentrate the peptides;and the peptides thereafter separated with micro LC and analyzed byon-line tandem mass spectrometry (Figeys et al., Anal Chem 1999; 71:2279-2287). Nucleotide and amino acid sequence homology searches wereconducted using the BLAST algorithm, and motif analyses performed usingthe program BLOCKS.

General transfection and analysis methods. Based on the human PAMPsequence, public databases (e.g., GenBank; www.ncbi.nlm.nih.gov) weresearched for homologous ESTs (SEQ ID NOs: 3-10), which were collectedinto a few contigs. These contigs all turned out to be correct, but didnot cover full-length mouse and D. melanogaster cDNAs.

Full length murine (SEQ ID NO: 15), human (SEQ ID NO: 13) and D.melanogaster (SEQ ID NO: 17) PAMP cDNAs were obtained usingoligonucleotides designed from partial cDNA/EST sequences in publicdatabases to screen appropriate cDNA libraries, for 5′RACE, and/or forRT-PCR experiments. A PAMP expression construct was generated byinserting human PAMP cDNA in-frame with the V5 epitope of pcDNA6(Invitrogen) at the C-terminus of PAMP. HEK293 cells with a stableexpression of PS1/PS2 and βAPP_(sw) were transiently transfected witheither V5-tagged PAMP or empty plasmid (mock transfection control).Duplicate experiments were performed by: (1) transient transfection ofV5-PAMP and βAPP₆₉₅ (or empty vector plus βAPP₆₉₅ as a mock transfectioncontrol) into murine embryonic fibroblasts stably infected with humanPS1 expressed from a retroviral vector construct (Clontech, CA); or (2)transient transfection of V5-PAMP (or an empty plasmid) into HEK293 celllines with a stable expression of the C-terminal 99 amino acids of βAPPwith a preceding artificial signal peptide (spC100-APP) together witheither wild type PS1, PS1-L392V, or PS1-D385A. Cells were lysed with aDigitonin lysis buffer or with 1% NP40, and the protein extracts weresubjected to gradient fraction, immunoprecipitation or direct Westernblotting as described (Yu G, et al., J Biol Chem 1998; 273:16470-16475). PS1 was immunodetected or immunoprecipitated withantibodies 14 or α-PS1-CTF; and PS2 was immunodetected orimmunoprecipitated with antibody DT2. FL-βAPP and its C-terminal α- andβ-secretase derivatives were detected using antibody 369.

Results

Isolation of PAMP. Immunoprecipitation of PS1 protein complexes,followed by SDS-PAGE with Coomassie Blue and silver staining, yieldedtwo intense bands in addition to presenilin holoprotein. These bandswere characterized by mass spectroscopy analysis. Mass spectroscopyanalysis revealed several armadillo repeat containing peptides,(previously known to functionally interact with presenilins (Yu G, etal., J Biol Chem 1998; 273-16470-16475.; Zhou J, et al., NeuroReport(Fast Track) 1997; 8: 2085-2090; Nishimura M, et al., Nature Med 1999;5: 164-169), and a novel peptide (AMP) which had a sequence identifiedto that predicted for an anonymous, partial cDNA (Genbank; Accession No.D87442). The cDNA sequence predicted an open reading frame of 709 aminoacids (SEQ ID NO: 14), which contains a putative N-terminal signalpeptide, a long N-terminal hydrophilic domain with sequence motifs forglycosylation, N-myristoylation and phosphorylation, a ˜20 residuehydrophobic putative transmembrane domain, and a short hydrophilicC-terminus of 20 residues (FIGS. 1A and 1B).

Orthologous PAMP proteins. The PAMP amino acid sequence had nosignificant homology to other proteins within available databases,except for a hypothetical C.elegans protein (www.ncbi.nih.gov; AccessionNo. Q23316) (p=2×10⁻²⁸; identity=22%; similarity=39%) (SEQ ID NO: 12)ascertained from a genomic DNA sequence (FIGS. 1A and 1B). In additionto strong primary amino acid sequence conservation, this C.elegansprotein has a very similar topology to human PAMP, suggesting that it isthe nematode orthologue of human PAMP.

In the absence of functional clues arising from homologies to otherknown proteins, the predicted amino acid sequences of the murine (SEQ IDNO: 16) and D.melanogaster (SEQ ID NO: 18) orthologues of PAMP werecloned and examined. The four orthologous PAMP proteins had a similartopology and significant sequence conservation near residues 306-360,419-458, and 625-662 of human PAMP (SEQ ID NO: 14) (FIGS. 1A and 1B).Motif analysis of these conserved domains revealed a weak similarity(strength=1046) between residues 625-641 (ARLARALSPAFELSQWS; SEQ ID NO:19) of mouse and human PAMP to cyclic nucleotide binding domains Whilethe putative transmembrane domain sequences were not highly conserved,all four orthologues contained a conserved serine residue within thishydrophobic domain. Finally, there were four conserved cysteine residuesin the—terminal hydrophilic domain (Cys₁₉₅, Cys₂₁₃, Cys₂₃₀, and Cys₂₄₈in human PAMP) which had a periodicity of 16-17 residues in theN-terminus, and may form a functional domain (e.g., a metal bindingdomain or disulfide bridges for stabilizing the tertiary structure ofPAMP/PAMP complexes).

Interaction of PAMP with presenilin 1. To confirm the authenticity ofthe PAMP:PS1 interaction, HEK293 cells were transiently transfected withPAMP cDNA (SEQ ID NO: 13) tagged at the 3′-end with a V5-epitope encodedfrom the pcDNA6 vector. The conditioned media were collected 20 hr aftertransient transfection with PAMP (or with empty vector), and the Aβ₄₀and Aβ₄₂ levels were measured by ELISA (Zhang L, et al., J Biol Chem1999; 274: 8966-8972.). In Western blots of lysates of these cells, theuse of anti-V5 (Invitrogen, CA) and enhanced chemiluminescence(Amersham) detected a V5-immunoreactive band of ˜110 kDa which wasreduced to ˜80 kDa following Endo H digestion (equivalent to the sizepredicted from the PAMP amino acid sequence), confirming the predictedglycosylation of PAMP. In addition, a series of about 7-10 kDa fragmentswere observed, which are predicted to contain the TM domain and shortC-terminus of PAS plus the 3 kDa V5-epitope. These C-terminalderivatives may be authentic cleavage products of full-length PAMP, or,alternatively, a proteolytic artifact arising from the attachment of theV5-epitope to the C-terminus of PAMP.

Reciprocal immunoprecipitation studies in cells expressing combinationsof transfected V5-tagged-PAMP, transfected wild type or mutant PS1,transfected wild type PS2, or endogenous presenilins, confirmed thePS1:PAMP interaction, and showed a similar interaction between PAMP andPS2. In contrast, immunoprecipitation of other ER-resident proteins(e.g., calnexin) failed to show any evidence of an interaction betweenthese proteins and PAMP. Glycerol velocity gradient analysis of thenative conformation of PAMP revealed that PAMP was co-eluted into thesame high molecular weight fractions as PS1 and PS2, indicating that itis an authentic component of the high molecular weight presenilinprotein complexes. These biochemical data were supported byimmunocytochemical studies, which showed that transfected PAMP andendogenous PS1 strongly co-localized in the ER and Golgi in MDCK caninekidney/epithelial cells (ATCC). Similar studies with PS2 confirmed thatPAMP also tightly associates with both endogenous PS2 in human brain andwith transfected PS2 in HEK293 cells.

The PAMP gene. Chromosomal locations and genetic map positions of themurine and human PAMPS were obtained, from public genetic andtranscriptional maps (World Wide Web (www) ncbi.nlm.nih.gov). The geneencoding PAMP is located on human chromosome 1 near the genetic markersD1S1595 and D1S2844. The 5′-end of the PAMP gene is embedded in the5′-end of the coatmer gene encoded on the opposite strand. The humanPAMP gene is close to a cluster of markers which have yielded positive,but sub-significant evidence for linkage to or association withAlzheimer Disease in two independent genome wide surveys (Kehoe P, etal. Hum Mol Genet 1999; 8: 237-245). The murine PAMP maps within a 700Kb interval of murine chromosome 1 which contains the gene defectassociated with Looptail phenotype in mice (Underhill D A, et al.,Genomics 1999; 55: 185-193). Mice heterozygous for Looptail showdevelopmental defects in dorsal axial structures including notochord,brain, spinal cord, and somites (Greene N D, et al., Mech Dev 1998; 73:59-72.), which are reminiscent of those observed in PS1-^(/−) mice (ShenJ, et al., Cell 1997; 89; 629-639; Wong P C, et al., Nature 1997;387:288-292). These observations suggest that the presenilin; PAMPcomplex may be involved in both βAPP and Notch processing.

C. elegans homolog of PAMP. The C. elegans homolog of PAMP correspondsto the aph-2 gene. Mutations in aph-2 have been identified in a screenfor mutants with phenotypes identical to embryonic mutant phenotypescaused by loss of glp-l activity, i.e., lack of an anterior pharynx,e.g. cDNA clone. The EST corresponding to aph-2, (cDNA clone yk477b8,kindly provided by Y. Kohara, National Institute of Genetics, Japan) wassequenced and the coding region (SEQ ID NO: 11) found to match exactlythe Genefinder prediction made by the C. elegans sequencing consortium(Genbank; Accession No. Z75714). Double stranded RNA interference (RNAi)confirmed the mutant phenotype of aph-2. Sense and antisense RNA weretranscribed in vitro from PCR product amplified from the phage yk477b8.After annealing equal quantities of sense and antisense products, thedsRNA product made was injected into L4 stage wild-type worms. Thechosen line of worms, designated lin-12(n302) (Greenwald and Seydoux,Nature 1990: 346:197-199, Greenwald, et al., Cell 1983: 34; 435-444) wasobtained from the Caenorhabditis Genetics Center. Injected animals weretransferred to fresh plates daily and the progeny scored at least 36hours alter injection for the embryonic lethal phenotype and 4-5 daysafter injection for the egg-laying phenotypes. Animals injected withdsRNA from yk477b8 template produced eggs that lacked an anteriorpharynx. These results support the notion that aph-2/PAMP contributes tocell interactions mediated by glp-l/Notch in the embryo.

Functional role for the PAMP: presenilin complexes in βAPP processing.To examine a functional role for the PAMP: presenilin complexes in βAPPprocessing, the interactions between PAMP, PS1, and βAPP, and itsderivatives were investigated. The cell lines used were transientlytransfected with V5-tagged PAMP, and stably expressing wild type βAPP₆₉₅in addition to wild type PS1, wild type PS2, PS1-L392V mutant, orPS1-D385A mutant. The PS1-L392V mutation is a pathogenic mutationassociated with familial AD (Sherrington R, et al., Nature 1995;375:754-760) and with increased secretion of Aβ₄₂ (Scheuner D, et al.Nature Med 1996; 2:864-870, Citron M, et al. Nature Med 1997; 3:67-72).The PS1-D385A mutation is a loss of function mutation associated withinhibition of PS1 endoproteolysis and a decrease in γ-secretase activity(Wolfe M S, et al., Nature 1999; 398: 513-517). The conditioned mediawere collected 20 hr after transient transfection with PAMP (or withempty vector), and the Aβ₄₀ and Aβ₄₂ levels were measured by ELISA(Zhang L, et al., J Biol Chem 1999; 274: 8966-8972). Analysis of Westernblots from these co-immunoprecipitation experiments revealed that PAMPholoprotein (and C-terminally tagged proteolytic fragments of PAMP)interacted in equivalent degrees with wild type PS1, wild type PS2,PS1-L392V mutant, and PS1-D385A mutant proteins. In addition, PAMPholoprotein and the C-terminal proteolytic fragments of PAMP alsoco-immunoprecipitated with the C-terminal proteolytic fragments of βAPPbut not βAPP holoprotein in lysates of cells expressing either βAPPholoprotein or just the C-terminal 99 amino acids of βAPP.Significantly, compared to cells expressing equivalent quantities ofwild type PS1, cell lines expressing pathogenic mutations of PS1 showedincreased amounts of C-terminal βAPP fragments co-immunoprecipitatingwith PAMP. Conversely, cell lines expressing the loss-of-functionPS1-D385A mutation showed greatly reduced amounts of C-terminal βAPPderivatives co-immunoprecipitating with PAMP despite the presence ofvery large amounts of C-terminal βAPP derivatives in these cells.

These results were confirmed in HEK293 cells over-expressing eitherβAPP_(Swedish) or the SpC99-βAPP cDNA. The latter encodes the C-terminal99 residues of βAPP (corresponding to the products of β-secretasecleavage) plus the βAPP signal peptide. The interaction of PAMP appearsmuch stronger with C99-βAPP than that with C83-βAPP. However, C83-βAPPis much less abundant in these cells. In fact, PAMP does interact withboth C99- and C83-βAPP stubs. Cumulatively, these results indicate thatPAMP likely interacts with the C-terminal derivatives of βAPP which arethe immediate precursors of Aβ and p3. However, of greater interest, thegenotype of the co-expressed PS1 molecule dynamically influenced theinteraction between PAMP and C99-/C83-βAPP stubs. Thus, more C-terminalβAPP fragments co-immunoprecipitated with PAMP in cells expressing theFAD-associated PS1-L392V mutation compared to cells expressing wild typePS1 (and equivalent quantities of nicastrin and C99-βAPP). Conversely,much less C-terminal βAPP derivatives co-immunoprecipitated with PAMP incell lines expressing the loss-of-function PS1-D385A mutation (despitethe presence of very large amounts of C-terminal βAPP derivatives inthese cells). These effects are more easily seen in cellsover-expressing the C99-βAPP construct. However, similar but lesspronounced differences were also observed in cells over-expressingfull-length βAPP_(Swedish). More importantly, the PS1-sequence-relateddifferences in the interaction of PAMP with C-terminal βAPP derivativeswere most evident within 24 hours of transient transfection of PAMP. By72 hours, the PS1-sequence-related differences were largely abolished.This dynamic change in the interaction of PAMP with C99/C83-βAPP was notdue to changes in the levels of PS1, C-terminal βAPP derivatives orPAMP. One interpretation of these results is that the presenilins may bedynamically involved in regulating or loading PAMP with the substratesof β-secretase.

Presenilin binding domains of PAMP. In transiently transfected cells (inwhich the 7-10 kDa C-terminal of PAMP can be detected), anti-PS1immunoprecipitation products contain both full length PAMP and the ˜7-10kDa C-terminal PAMP fragments. Similarly, in these cells,immunoprecipitation with antibodies to the C-terminus of βAPP (Ab369)also renders C-terminal nicastrin epitopes. The TM domain of PAMP is nothighly conserved in evolution. These results suggest that theC99-/C83-βAPP and PS1/PS2-binding domain(s) of PAMP are in the TM domainor C-terminus.

Discussion

The above results indicate that PAMP is a component of the PS1 and PS2intracellular complexes. The observations that PAMP also binds to theC-terminal fragment of βAPP (arising from α- and β-secretase cleavage offull length βAPP), that the degree of binding of these fragments to PAMPis modulated by mutations in PS1, and that the direction of thismodulation is congruent with the effects of each mutant of Aβ production(i.e., the pathogenic L392V mutation increases binding to PAMP andincreases Aβ₄₂ production whereas the D385A mutation has the oppositeeffects) strongly argues that PAMP is part of a functional complexinvolved in processing of C-terminal βAPP derivatives. Similarly, theobservation that inhibition of PAMP expression in C. elegans leads to aphenotype similar to that of glp/Notch loss of function, argues thatPAMP, like PS1 and PS2, is also a functional component of the pathwaysinvolved in processing of Notch. This conclusion is strengthened by thefact that the murine PAMP gene maps within a 700 kb interval on murinechromosome 1 which carries the Looptail mutant, and is thus likely to bethe site of the Looptail mutation. Looptail has a number of phenotypicsimilarities to those of Notch and PS1 knockouts in mice. BecauseLooptail is a model of human spinal cord malformations including spinabifida, PAMP biology may also provide some useful insights into thisneurological developmental defect as well.

At the current time the exact role of PAMP in thepresenilin-complex-mediated processing of βAPP and Notch-like moleculesis not fully defined. Inspection of the primary amino acid sequence ofPAMP does not reveal very strong homologies to known proteases. However,the recombinant expression systems of the invention permit evaluation ofthree-dimensional structure of PAMP; it is possible that PAMP itself hasa protease activity. However, it is currently more plausible that PAMPplays another role in βAPP and Notch processing. Thus, PAMP may beinvolved in the function of PS1 and PS2 complexes by binding substratesfor γ-secretase. The efficacy of this binding is clearly modulated byPS1 mutations in a direction which is commensurate with the effect ofthese mutations on γ-secretase activity. Alternatively, PAMP may have aregulatory role on the activity of the presenilin complexes. This isconsistent with the observation that residues 625-641 of human andmurine PAMP contain a motif similar to cyclic nucleotide binding domainsof several other unrelated proteins.

Regardless of its precise role, it is clear that PAMP and PS1 both playimportant roles in γ-secretase mediated processing of βAPP. Hence,knowledge of PAMP and its biology will now serve as a target for effortsto manipulate the function of the presenilin complexes in patients withAlzheimer Disease and related disorders, patients with malignancies (inwhich the presenilins have been implicated by virtue of a role inprogrammed cell death), and in disorders of development especially ofthe spinal cord and brain (in view of the known effects of PS1 knockoutand the strong likelihood that PAMP is the site of Looptail mutations inmice). In particular, knowledge of the domains of PAMP involved inbinding presenilins and βAPP derivatives (which currently appears to belocated within the C-terminal transmembrane and hydrophilic domains ofPAMP) and the identification of putative ligands interacting with theconserved domains at the hydrophilic N-terminus of PAMP willconsiderably expedite this goal.

We have found that the strength of the interaction between PAMP and theC-terminal fragments of βAPP (which is the precursor Aβ) is determinedby the genotype at PS1. Thus, clinical mutations in PS1 which causeAlzheimer Disease and an increase in the production of Aβ₄₂ areassociated with increased binding of the C-terminal fragments of βAPP toPAMP. Conversely, loss of function mutations in PS1 (Asp385Ala) whichinhibit γ-secretase cleavage of C-terminal fragments of βAPP, areassociated with abolition of the interaction between PAMP and theC-terminal fragments of βAPP.

Finally, the apparent C-terminal proteolytic derivatives of PAMP couldeither be authentic, or simply artefacts due to the V-5 tag. If they areauthentic, this observation raises the possibility that PAMP may undergopost-translational processing events which are potentially similar At tothose of βAPP and/or Notch. Three observations support our discovery ofPAMP. First, in contrast to βAPP and Notch, which are not majorconstituents of the high molecular weight presenilin complexes, andwhich can only be inconsistently shown to be directly associated withPS1/PS2, PAMP is a major stoichiometric component of the presenilincomplexes. Second, PAMP selectively interacts only with C-terminalderivatives of βAPP which are substrates for γ-secretase cleavage, andthis interaction is modulated by PS1 mutations in a way which reflectsthe functional consequences of these PS1 mutations. Third, inhibition ofPAMP expression in C. elegans leads to a disease phenotype likely to bein the glp/Notch signaling pathway.

Example 2 PAMP Mutants Useful for Studies on Alzheimer's Disease

Site-directed mutagenesis was used to generate the following artificialmutations in PAMP:

Cys: PAMP_(C230A) in the 4 conserved cystine motif

DYIGS: PAMP_(D336A/Y337A) in the central conserved region

D369L: PAMP_(Δ312-369) in the central conserved region

D340X: PAMP_(Δ312-340) in the central conserved region

YDT: PAMP_(D458A) in the putative ‘aspartyl protease’ DTA site

SPAF: PAMP_(P633A/F635A) in the SPAF motif

TM: PAMP_(S683A) in the TM domain

C3D: PAMP_(Δ630-668) in the conserved region adjacent to the TM domain

To further examine the role of nicastrin in βAPP processing, we insertedPAMP cDNAs, harboring the above mutations as well as normal/wild typePAMP (PAMP_(wt)) cDNA and the cDNA for an unrelated protein (LacZ), inframe into pcDNA6 vectors. A series of HEK293 cell lines stablyexpressing endogenous PS1, βAPP_(Swedish) and either wild type nicastrinor nicastrin constructs in which various conserved domains had beenmutated or deleted, were then created by transfection. PAMP expressingcells were selected with lasticidin to generate stable cell lines.Conditioned media from these cell lines were collected after 6-24 hoursand Aβ₄₀ and Aβ₄₂ were measured by ELISA.

In the PAMP_(D336A/Y337A) mutant, both Aβ₄₀ and Aβ₄₂ levels wereincreased, and there was also a 68% increase in Aβ₄₂/Aβ₄₀ ratio which isvery similar to that observed in clinical mutations in APP, PS1, andPS2, associated with early onset Alzheimer Disease. The Aβ₄₂/Aβ₄₀ ratiowas also increased in one cell line expressing the PAMP_(C230A) mutant.

In contrast, both the total Aβ₄₂ and Aβ₄₀ levels and the Aβ₄₂/Aβ₄₀ ratiowere massively reduced (to only 18% of the control) in thePAMP_(Δ312-369) mutant. A similar but less profound reduction of boththe total Aβ₄₂ and Aβ₄₀ levels and the Aβ₄₂/Aβ₄₀ ratio was observed inthe conditioned medium from the PAMP_(Δ312-340) cell lines.

There is no apparent difference in Aβ₄₂ or Aβ₄₀ levels, or in theAβ₄₂/Aβ₄₀ ratio, when the PAMP_(wt), PAMP_(D458A), PAMP_(Δ630-668),PAMP_(P633A/F635A), and PAMP_(S683A) cells were compared to controllines (expressing LacZ, or empty vector).

Thus, certain PAMP mutants cause biochemical changes similar to thoseinduced by mutations in the βAPP, PS1, and PS2 genes which give rise toAlzheimer Disease. These artificial PAMP mutations can therefore be usedto generate cellular and other model systems to design treatments andpreventions for Alzheimer Disease related disorders. These mutationsalso show that PAMP is involved in the pathogenesis of AD, and mayprovide information for the design of specific molecular diagnostics ortherapeutics.

When compared to mock-transfected or LacZ transfected cells,overexpression of wild type PAMP, and overexpression of most PAMPmutation- or deletion-constructs had no significant effect on Aβsecretion. However, missense mutation of the conserved DYIGS motif toAAIGS (residues 336-340 of human PAMP) caused a significant increase inAβ₄₂ secretion, a smaller increase in Aβ₄₀ secretion, and an increase inthe Aβ₄₂/Aβ₄₀ ratio (p<0.001; Table 2). This increase in Aβ₄₂ productionwas equivalent to that of FAD-related missense mutations in PS1.Conversely, deletion of the DYIGS domain in two independent constructs(PAMP_(Δ312-369) and PAMP_(Δ312-340)) caused a significant reduction inboth Aβ₄₂ and Aβ₄₀ secretion which was more profound in PAMP_(Δ312-369)cells than in PAMP_(Δ312-340) cells (Table 2). The magnitude of thereduction in Aβ secretion in PAMP_(Δ312-369) cells was equivalent tothat observed with the PS1-D385A loss-of-function mutation. Somewhatunexpectedly, and in contrast to PS1^(−/−) and PS1-D385A cells, thereduction in Aβ secretion in NCT_(Δ312-369) and NCT_(Δ312-340) cells wasnot accompanied by the expected accumulation of C99- and C83-βAPP stubs.Since there was no consistent change in the levels of soluble βAPP(βAPP_(a)) in the conditioned medium of any of the PAMP mutant cells,the most probable explanation for this result is that C99- and C83-βAPPstubs which do not enter the PAMP:presenilin complex for γ-secretasecleavage to Aβ may be degraded by other pathways.

The effects of PAMP mutations on Aβ secretion were not due to trivialexplanations such as differences in the levels of PAMP, βAPPholoprotein, or PS1/PS2. None of these mutations caused any consistent,detectable change in the amount of APP_(a) in conditioned medium or inthe amount of C99/C83-βAPP that could be co-immunoprecipitated withPAMP. However, both the PAMP_(Δ312-369) mutant and the PAMP_(Δ312-340)deletion mutant significantly reduced the amount of PS1 which could beco-immunoprecipitated with PAMP. Interestingly, the reduction inefficiency of binding to PS1 was proportional to the reduction in Aβsecretion induced by each deletion mutant. Multiple mechanismsunderlying the effect of mutations in the first conserved domain canexplain these results. This domain contains no obvious functional motifs(e.g., for glycosylation etc.), nor does it have significant sequencehomology to other known proteins. Consequently, the three functionallyactive PAMP mutations either affect a presenilin-binding domain in PAMP,or affect a specific regulatory domain of PAMP which modulates bothdirect binding of PAMP to PS1 and the subsequent γ-secretase-mediatedcleavage of PAMP-bound C99- and C83-βAPP stubs.

TABLE 2 Normalized Transfection Normalized Aβ₄₂ Aβ₄₀ Aβ₄₂/Aβ₄₀ RatioMock (LacZ/empty 1.0 1.0 1.0 vector) wild type PAMP 1.03 ± 0.09 1.05 ±0.07 0.99 ± 0.07 D336A/Y337A 3.09 ± 0.59 1.61 ± 0.19 1.81 ± 0.15 (p <0.001) (p = 0.001) (p < 0.001) PAMP_(Δ312-369) 0.05 ± 0.04 0.31 ± 0.060.09 ± 0.05 (p < 0.001) (p < 0.001) (p < 0.001) PAMP_(Δ312-340) 0.33 ±0.04 0.55 ± 0.04 0.59 ± 0.06 (p = 0.002) (p = 0.001) (p = 0.003)

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that values are approximate, and areprovided for description.

Patents, patent applications, and publications are cited throughout thisapplication, the disclosures of which are incorporated herein byreference in their entireties.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 19 <210> SEQ ID NO 1 <211>LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 1 This Sequence isintentionally skipped <210> SEQ ID NO 2 <211> LENGTH: <212> TYPE: <213>ORGANISM: <400> SEQUENCE: 2 This Sequence is intentionally skipped <210>SEQ ID NO 3 <211> LENGTH: 422 <212> TYPE: DNA <213> ORGANISM: mouse<400> SEQUENCE: 3 cccagcggag aggcaagatg gctacgacta ggggcggctc tgggcctgacccaggaagtc 60 ggggtcttct tcttctgtct ttttccgtgg tactggcagg attgtgtgggggaaactcag 120 tggagaggaa aatctacatt cccttaaata aaacagctcc ttgtgtccgcctgctcaacg 180 ccactcatca gattggctgc cagtcttcaa ttagtgggga tacaggggttatccatgtag 240 tggagaaaga agaagactga agtgggtgtt gacgatggcc ccaacccccttacatggtct 300 gctggaggga agtcttcaca gagatgtaat ggagaagctg aggacaacagtagatcctgg 360 tcttgccgtg attagcagcc actcacttaa gtttctctag tgtgagtgccaatgatgggt 420 tt 422 <210> SEQ ID NO 4 <211> LENGTH: 473 <212> TYPE:DNA <213> ORGANISM: unknown <220> FEATURE: <223> OTHER INFORMATION: ESTfrom unknown organism <400> SEQUENCE: 4 tggagaggaa aatctacatt cccttaaataaaacagctcc ttgtgtccgc ctgctcaacg 60 ccactcatca gattggctgc cagtcttcaattagtgggga tacaggggtt atccatgtag 120 tggagaaaga agaagacctg aagtgggtgttgaccgatgg ccccaacccc ccttacatgg 180 ttctgctgga gggcaagctc ttcaccagagatgtaatgga gaagctgaag ggaacaacca 240 gtagaatcgc tggtcttgcc gtgactctagccaagcccaa ctcaacttca agcttctctc 300 ctagtgtgca gtgcccaaat gatgggtttggtaattactc caactcctac gggccagagt 360 ttgctcactg gaagaaaaca ctgtggaatgaactcggcaa aggcttggct tatgaagacc 420 ttagtttccc caatcttcct cctggagatgaggaccgaaa caaggtcatc aag 473 <210> SEQ ID NO 5 <211> LENGTH: <212>TYPE: <211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 5 ThisSequence is intentionally skipped <210> SEQ ID NO 6 <211> LENGTH: 463<212> TYPE: DNA <213> ORGANISM: unknown <220> FEATURE: <223> OTHERINFORMATION: EST from unknown organism <400> SEQUENCE: 6 gggctcgaaacatctctggc gtggtcctgg ctgaccactc tggctccttc cacaatcggt 60 attaccagagcatttatgac acggctgaga acattaatgt gacctatcct gagtggcaga 120 gccatgaagaggacctcaac tttgtgacag acactgccaa ggcactggcg aatgtggcca 180 cagtgctggcgcgtgcactg tatgagcttg caggaggaac caacttcagc agctccatcc 240 aggctgatccccagacagtt acacgtctgc tctatgggtt cctggttaga gctaacaact 300 catggtttcagtcgatcctg aaacatgacc taaggtccta tttggatgac aggcctcttc 360 aacactacatcgccgtctcc agccctacca acacgactta cgttgtgcag tacgccttgg 420 caaacctgactgggcaaggc gaccaacctc acccgagagc agt 463 <210> SEQ ID NO 7 <211> LENGTH:481 <212> TYPE: DNA <213> ORGANISM: unknown <220> FEATURE: <223> OTHERINFORMATION: EST from unknown organism <400> SEQUENCE: 7 gaggacctcaactttgtgac agacactgcc aaggcactgg cgaatgtggc cacagtgctg 60 gcgcgtgcactgtatgagct tgcaggagga accaacttca gcagctccat ccaggctgat 120 ccccagacagttacacgtct gctctatggg ttcctggtta gagctaacaa ctcatggttt 180 cagtcgatcttgaaacatga cctaaggtcc tatttggatg acaggcctct tcaacactac 240 atcgccgtctccagccctac caacacgact tacgttgtgc agtacgcctt ggcaaacctg 300 actggcaaggcgaccaacct cacccgagag cagtgccagg atccaagtaa agtcccaaat 360 gagagcaaggatttatatga atactcgtgg gtacaaggcc cttggaattc caacaggaca 420 gagaggctcccacagtgtgt gcgctcacag tgcgactggc aagggcttgt ccctgccttt 480 g 481 <210>SEQ ID NO 8 <211> LENGTH: 398 <212> TYPE: DNA <213> ORGANISM: unknown<220> FEATURE: <223> OTHER INFORMATION: EST from unknown organism <400>SEQUENCE: 8 agagctaaca actcatggtt tcagtcgatc ttgaaacatg acctaaggcctatttggatg 60 acaggcctct tcaacactac atcgccgtct ccagccctac caacacgacttacgttgtgc 120 agtacgcctt ggaaacctga ctggcaaggc gaccaacctc acccgagagcagtgccagga 180 tccaagtaaa gtcccaaatg agagcaagga tttatatgaa tactcgtgggtacaaggccc 240 ttggaattcc aacaggacag agaggctccc acagtgtgtg cgctccacagtgcgactggc 300 cagggccttg tcccctgcct ttgaactgag tcagtggagc tccacagaatactctacgtg 360 ggcggagagc cgctggaaag acatccaagc tcggatat 398 <210> SEQID NO 9 <211> LENGTH: 172 <212> TYPE: DNA <213> ORGANISM: unknown <220>FEATURE: <223> OTHER INFORMATION: EST from unknown organism <400>SEQUENCE: 9 tgtgcgctcc acagtgcgac tggccagggc gttgtcacct gcctttgaactgagtcagtg 60 gagctccaca gaatactcta cgtgggcgga gagcgcgtgg aaagacatcccagctcggat 120 attcctaatt gccagcaaag agcttgagtt catcacgctg atcgtgggct tc172 <210> SEQ ID NO 10 <211> LENGTH: 425 <212> TYPE: DNA <213> ORGANISM:unknown <220> FEATURE: <223> OTHER INFORMATION: EST from unknownorganism <400> SEQUENCE: 10 tttttttttt ttttttgtat tgcataattt taatgaaacttgctatttat atacttacaa 60 aaaaaaaaaa aaaggaaaaa accccaacaa aaatagataattatagttta ataataaaaa 120 gtacaactga gcactgtggg ctggaggtgg gatacccacttaacagcgtg cccacactaa 180 catgccatct gcacacctgg agaaaggaca gtgggaaagagacactggct cagccaggga 240 atccatttct tccctaaggg ttcagggtag ttgaatgcagatgcacaatc tttcacaccc 300 tcttcctggt gcagcaggtg gctgaatatg ggggaggggtgtcgggtgac agtggagtca 360 gagggcagta cagggcagga tggaaggaca gaaggtatcccgagaaaggg cagaggaggg 420 tgggt 425 <210> SEQ ID NO 11 <211> LENGTH:4560 <212> TYPE: DNA <213> ORGANISM: C. Elegans <400> SEQUENCE: 11attaagaacg aatgagtcga tagaagcact gaaaaatgaa gaaatggcta gttatagtat 60taattatcgc tggaatacga tgcgacggat tttcggatca agttttccga actctgttca 120ttggagaagg aaatgcgtgc tatagaactt ttaataaaac gcacgaattc ggttgtcaag 180gtaaaaattg aatgatttca aataattaca tataaaaaaa tattgcactg ttttttcatt 240attttcattg aaaattagtg tcaaaatatg tataaatcaa tatttatctg aaaataactg 300gaaatataga gaaagtgctc caaaatggcc aaaacgttgt caattgccga agacgatact 360ctataataaa cggcaattgg caactttcgg gctgtttttc aacactgttc aatttgtcag 420atgaaaataa ttttattttc agttaactca agtgattttc tatattgtgg cagtgaaaaa 480aattcatagg ccattttgta gaattgccga aaataactcc acctctgaat tacatgcatt 540ttcactagaa aatatcattt acatacattt taatttataa atatccagta tttatttatt 600ttcttaaact cattttcaag aaaaatattt tcagctaatc gagaaaacga gaatggccta 660attgttcgaa tcgacaaaca ggaagacttc aaaaatctcg attcttgctg gaattcattt 720tatcccaaat attccgggaa atattgggca cttctcccag tcaatttgat tcgtcgtgat 780acaatttctc aattgaaatc atcgaaatgt ctttctggaa tagtattata taatagtgga 840gaatctattc atccaggaga tgaatcaaca gcagcttcac atgatgcaga atgtccaaat 900gctgcaagtg attattatct tcaagataaa aatgaagaat attgtgaaag aaagattaat 960tctcggggtg ctataacacg agatggatta atgaaaattg attggcggat acaaatggta 1020tttattgata attcaactga tttggaaatc attgagaaat gttattcaat gttcaataaa 1080ccaaaagaag atggttcatc tggatatcca tattgtggaa tgagctttcg tttggctaat 1140atggcggctg gaaattcaga aatttgctat cggcgtggaa aaaacgatgc aaagctgttt 1200cagatgaata ttgatagcgg gtaggttttt aaattttaag cagttaaaag aggtgaattt 1260ttgcattatt aaatgcagaa tagaccgtaa atattgcatg atgagatgta tttcatgata 1320atattcttta agaaaataaa tttgaaaatt tcataggaaa ataaacaaaa ttttgctaaa 1380cttcatagtt tggcatttct tatctcgttt tttgttaatt taggggattt tttagtcaat 1440aattgcaccg attccatgta tctctttttt tcgaatgata ttgtacctat atgccagacg 1500agctataatt tcctaatttt aaaaaataaa ttgtccaact caatgcctca atagttgaag 1560ttttccagag atgctcctca actctgtggt gcaatgcaca gtgacaatat atttgcattt 1620ccaactccaa ttccaacttc tccaacaaat gagacaataa tcacgagcaa atatatgatg 1680gtaactgctc gaatggacag ttttggaatg attccagaga tttctgttgg cgaagtatcc 1740gtactaactt caattatttc tgtactcgca gcagctcgat caatgggaac acagatcgaa 1800aaatggcaga aagcatcgaa tacttcgaat cgtaatgttt tctttgcttt tttcaatggt 1860gaatcgttgg attatattgg aagtggtgcg gctgcgtatc agatggagta agttggaaaa 1920tttaatttaa aaaacgttct agaactagta actgatcaaa aaaatttccc tattaacata 1980aaatggccca aaaattccta aaaatttcaa aatttcaaaa aaaaaaatag ttcgggcaaa 2040aaacataatt ctagctgaaa cctcaaattt ggcaagcttt tcaggctcgt aacatatttt 2100tggaagtcgt caatcaaaaa ataattcagt tttattcatt tatgataatt aattaaaatt 2160ttccaacatt gtttgaaaat ttttataatg atatttggtc attttaccat aattggaatg 2220gttttcaatt attttcccac tcttccttta gagaaaaaat atatttgtct tcagaaatgg 2280aaagttccca caaatgattc gctctgatcg aacacacatt catccaattc gcccgaatga 2340gttagattat atactggaag tacaacaaat tggagttgct aaaggacgaa aatattatgt 2400acacgttgat ggagaacgat atcaacagaa taagacacag acagatcgag ttattgatcg 2460aattgaacga ggtcttcgta gtcatgcttt tgatcttgaa aaaccatctg gaagtggaga 2520taggtgggtg catcgaaaat agtttttttt ttcaagaaca tacagaaaac gaaaagcttt 2580taaagcattt tctttaaaaa ttaaaacaat ttgagcatat gtaaactaca attccgagtg 2640tcgtttttcg aaaaaagtct aaaattaaaa aaaagcttat cgctcactat ttttcgaaaa 2700taaggtattt ttcctttaat aaaggcaaac gaaaaatctt cagccatgga taggtgaatt 2760atagaaataa ttttcaaaaa ttttcctttt tcagagttcc acccgcaagt tggcactcgt 2820ttgccaaggc tgatgctcac gttcaatcag ttctccttgc accatatggt aaagaatatg 2880aatatcaacg agttaattca attttggata aaaatgagtg gacagaagac gaacgagaga 2940aagcaattca agagattgaa gctgtttcta ctgctattct ggcagcagcc gctgattatg 3000ttggagttga aactgatgaa gttgttgcaa aagttgataa aaaattggta tgtattcttt 3060ttttttttaa ttttaaaact ttcagcgaca atttagatgt tttattgttg aatttgaaat 3120ttgcagtatt tttaaatact taaaacaaaa tccctgatga cgcagcgatt catcgctgta 3180ttttctaatt gctgaaattg aattccatat atatggaata tttcatatct ttacatataa 3240acgttttttt ttttcagata accactatat tcgattgtct catcacttcc aatttctggt 3300tcgactgtga ttttatgcaa aaactcgatg gcggtcgcta ccacaagctg tttaattcct 3360acggttttaa tcaaaaatca acatatattt caatggaatc ccatactgca ttccctaccg 3420tactccattg gttaactatt ttcgctttgg gtagtgacaa agaaacattg aatgtgaaaa 3480gtgaaaagag ctgctcacat cttggtcaat ttcaagcggt gagtttttat tttaaacgaa 3540tatcaaataa ttaaaatagt tttccgccag tttcagatgt atacctacac gtggcaaccg 3600aatccgtaca ccggaaattt cagttgtctg aaatctgcaa ttgttaaaaa agtaatggtt 3660tcgccggctg tagattctca aacacccgaa gaagaaatga acacgagata ttcaacatgg 3720atggaatcag tttatattat tgaatctgtg aatttatatt tgatggaaga tgcttcattt 3780gaatatacaa tgattctgat tgcggttatt tctgctttat tatcaatctt tgcagttggt 3840tagttttttt ttcaaaaaaa aaattacaaa aataaatcac aagctttcga gctttctcgt 3900attcgaaaat gaaggagttt cgcattaaag aaaactagat tttgaatcag tttttctaat 3960ctttagagaa attatactca catttgatgc ccagaaaagt ttgcgacttt tgagccaaaa 4020gcacggtgcc aggtctcgac acgaaaaatt tatattaatt gaaaatatgt ttgcgccttt 4080aaatggtact gtattttcga attctcattg ctggcgattt aaaaaaatgc attttttaaa 4140tccataaaag ttgagaaaaa tcgatgaaaa attgcacaga aatgagtgca agaaattaca 4200gtattcttta aaggcgcaca ccttttcgca tttcacaaaa tttcatcgtg tcgataccgg 4260gtaccgtatt ttggaggcaa aaatcgcaaa atctcgcgtc tggataatat cgtttatcgt 4320ttattgaagg aagtttttaa aaataagaaa aattgacagc tgcgagaaat tatgcataat 4380ttataaaaca ataaaaattt tttttttcag gtcgctgttc tgaaacaaca tttatcgttg 4440acgagggaga accagcagcg gaaggaggag aacctcttta acaaattatt ctcttcaaca 4500atgtatcata aattgattaa tttatttaat atttatattc gaaaaaatgt tcccattttt 4560<210> SEQ ID NO 12 <211> LENGTH: 721 <212> TYPE: PRT <213> ORGANISM: C.Elegans <400> SEQUENCE: 12 Met Lys Lys Trp Leu Val Ile Val Leu Ile IleAla Gly Ile Arg Cys 1 5 10 15 Asp Gly Phe Ser Asp Gln Val Phe Arg ThrLeu Phe Ile Gly Glu Gly 20 25 30 Asn Ala Cys Tyr Arg Thr Phe Asn Lys ThrHis Glu Phe Gly Cys Gln 35 40 45 Ala Asn Arg Glu Asn Glu Asn Gly Leu IleVal Arg Ile Asp Lys Gln 50 55 60 Glu Asp Phe Lys Asn Leu Asp Ser Cys TrpAsn Ser Phe Tyr Pro Lys 65 70 75 80 Tyr Ser Gly Lys Tyr Trp Ala Leu LeuPro Val Asn Leu Ile Arg Arg 85 90 95 Asp Thr Ile Ser Gln Leu Lys Ser SerLys Cys Leu Ser Gly Ile Val 100 105 110 Leu Tyr Asn Ser Gly Glu Ser IleHis Pro Gly Asp Glu Ser Thr Ala 115 120 125 Ala Ser His Asp Ala Glu CysPro Asn Ala Ala Ser Asp Tyr Tyr Leu 130 135 140 Gln Asp Lys Asn Glu GluTyr Cys Glu Arg Lys Ile Asn Ser Arg Gly 145 150 155 160 Ala Ile Thr ArgAsp Gly Leu Met Lys Ile Asp Trp Arg Ile Gln Met 165 170 175 Val Phe IleAsp Asn Ser Thr Asp Leu Glu Ile Ile Glu Lys Cys Tyr 180 185 190 Ser MetPhe Asn Lys Pro Lys Glu Asp Gly Ser Ser Gly Tyr Pro Tyr 195 200 205 CysGly Met Ser Phe Arg Leu Ala Asn Met Ala Ala Gly Asn Ser Glu 210 215 220Ile Cys Tyr Arg Arg Gly Lys Asn Asp Ala Lys Leu Phe Gln Met Asn 225 230235 240 Ile Asp Ser Gly Asp Ala Pro Gln Leu Cys Gly Ala Met His Ser Asp245 250 255 Asn Ile Phe Ala Phe Pro Thr Pro Ile Pro Thr Ser Pro Thr AsnGlu 260 265 270 Thr Ile Ile Thr Ser Lys Tyr Met Met Val Thr Ala Arg MetAsp Ser 275 280 285 Phe Gly Met Ile Pro Glu Ile Ser Val Gly Glu Val SerVal Leu Thr 290 295 300 Ser Ile Ile Ser Val Leu Ala Ala Ala Arg Ser MetGly Thr Gln Ile 305 310 315 320 Glu Lys Trp Gln Lys Ala Ser Asn Thr SerAsn Arg Asn Val Phe Phe 325 330 335 Ala Phe Phe Asn Gly Glu Ser Leu AspTyr Ile Gly Ser Gly Ala Ala 340 345 350 Ala Tyr Gln Met Glu Asn Gly LysPhe Pro Gln Met Ile Arg Ser Asp 355 360 365 Arg Thr His Ile His Pro IleArg Pro Asn Glu Leu Asp Tyr Ile Leu 370 375 380 Glu Val Gln Gln Ile GlyVal Ala Lys Gly Arg Lys Tyr Tyr Val His 385 390 395 400 Val Asp Gly GluArg Tyr Gln Gln Asn Lys Thr Gln Thr Asp Arg Val 405 410 415 Ile Asp ArgIle Glu Arg Gly Leu Arg Ser His Ala Phe Asp Leu Glu 420 425 430 Lys ProSer Gly Ser Gly Asp Arg Val Pro Pro Ala Ser Trp His Ser 435 440 445 PheAla Lys Ala Asp Ala His Val Gln Ser Val Leu Leu Ala Pro Tyr 450 455 460Gly Lys Glu Tyr Glu Tyr Gln Arg Val Asn Ser Ile Leu Asp Lys Asn 465 470475 480 Glu Trp Thr Glu Asp Glu Arg Glu Lys Ala Ile Gln Glu Ile Glu Ala485 490 495 Val Ser Thr Ala Ile Leu Ala Ala Ala Ala Asp Tyr Val Gly ValGlu 500 505 510 Thr Asp Glu Val Val Ala Lys Val Asp Lys Lys Leu Ile ThrThr Ile 515 520 525 Phe Asp Cys Leu Ile Thr Ser Asn Phe Trp Phe Asp CysAsp Phe Met 530 535 540 Gln Lys Leu Asp Gly Gly Arg Tyr His Lys Leu PheAsn Ser Tyr Gly 545 550 555 560 Phe Asn Gln Lys Ser Thr Tyr Ile Ser MetGlu Ser His Thr Ala Phe 565 570 575 Pro Thr Val Leu His Trp Leu Thr IlePhe Ala Leu Gly Ser Asp Lys 580 585 590 Glu Thr Leu Asn Val Lys Ser GluLys Ser Cys Ser His Leu Gly Gln 595 600 605 Phe Gln Ala Met Tyr Thr TyrThr Trp Gln Pro Asn Pro Tyr Thr Gly 610 615 620 Asn Phe Ser Cys Leu LysSer Ala Ile Val Lys Lys Val Met Val Ser 625 630 635 640 Pro Ala Val AspSer Gln Thr Pro Glu Glu Glu Met Asn Thr Arg Tyr 645 650 655 Ser Thr TrpMet Glu Ser Val Tyr Ile Ile Glu Ser Val Asn Leu Tyr 660 665 670 Leu MetGlu Asp Ala Ser Phe Glu Tyr Thr Met Ile Leu Ile Ala Val 675 680 685 IleSer Ala Leu Leu Ser Ile Phe Ala Val Gly Arg Cys Ser Glu Thr 690 695 700Thr Phe Ile Val Asp Glu Gly Glu Pro Ala Ala Glu Gly Gly Glu Pro 705 710715 720 Leu <210> SEQ ID NO 13 <211> LENGTH: 2949 <212> TYPE: DNA <213>ORGANISM: human <400> SEQUENCE: 13 tctgcagaat tcggcttgcg cctggaaacacgaacttccg gtctcttagg ctccgggcca 60 cagagacggt gtcagtggta gcctagagaggccgctaaca gacaggagcc gaacgggggc 120 ttccgctcag cagagaggca agatggctacggcagggggt ggctctgggg ctgacccggg 180 aagtcggggt ctccttcgcc ttctgtctttctgcgtccta ctagcaggtt tgtgcagggg 240 aaactcagtg gagaggaaga tatatatccccttaaataaa acagctccct gtgttcgcct 300 gctcaacgcc actcatcaga ttggctgccagtcttcaatt agtggagaca caggggttat 360 ccacgtagta gagaaagagg aggacctacagtgggtattg actgatggcc ccaacccccc 420 ttacatggtt ctgctggaga gcaagcattttaccagggat ttaatggaga agctgaaagg 480 gagaaccagc cgaattgctg gtcttgcagtgtccttgacc aagcccagtc ctgcctcagg 540 cttctctcct agtgtacagt gcccaaatgatgggtttggt gtttactcca attcctatgg 600 gccagagttt gctcactgca gagaaatacagtggaattcg ctgggcaatg gtttggctta 660 tgaagacttt agtttcccca tctttcttcttgaagatgaa aatgaaacca aagtcatcaa 720 gcagtgctat caagatcaca acctgagtcagaatggctca gcaccaacct tcccactatg 780 tgccatgcag ctcttttcac acatgcatgctgtcatcagc actgccacct gcatgcggcg 840 cagctccatc caaagcacct tcagcatcaacccagaaatc gtctgtgacc ccctgtctga 900 ttacaatgtg tggagcatgc taaagcctataaatacaact gggacattaa agcctgacga 960 cagggttgtg gttgctgcca cccggctggatagtcgttcc tttttctgga atgtggcccc 1020 aggggctgaa agcgcagtgg cttcctttgtcacccagctg gctgctgctg aagctttgca 1080 aaaggcacct gatgtgacca ccctgccccgcaatgtcatg tttgtcttct ttcaagggga 1140 aacttttgac tacattggca gctcgaggatggtctacgat atggagaagg gcaagtttcc 1200 cgtgcagtta gagaatgttg actcatttgtggagctggga caggtggcct taagaacttc 1260 attagagctt tggatgcaca cagatcctgtttctcagaaa aatgagtctg tacggaacca 1320 ggtggaggat ctcctggcca cattggagaagagtggtgct ggtgtccctg ctgtcatcct 1380 caggaggcca aatcagtccc agcctctcccaccatcttcc ctgcagcgat ttcttcgagc 1440 tcgaaacatc tctggcgttg ttctggctgaccactctggt gccttccata acaaatatta 1500 ccagagtatt tacgacactg ctgagaacattaatgtgagc tatcccgaat ggctgagccc 1560 tgaagaggac ctgaactttg taacagacactgccaaggcc ctggcagatg tggccacggt 1620 gctgggacgt gctctgtatg agcttgcaggaggaaccaac ttcagcgaca cagttcaggc 1680 tgatccccaa acggttaccc gcctgctctatgggttcctg attaaagcca acaactcatg 1740 gttccagtct atcctcaggc aggacctaaggtcctacttg ggtgacgggc ctcttcaaca 1800 ttacatcgct gtctccagcc ccaccaacaccacttatgtt gtacagtatg ccttggcaaa 1860 tttgactggc acagtggtca acctcacccgagagcagtgc caggatccaa gtaaagtccc 1920 aagtgaaaac aaggatctgt atgagtactcatgggtccag ggccctttgc attctaatga 1980 gacggaccga ctcccccggt gtgtgcgttctactgcacga ttagccaggg ccttgtctcc 2040 tgcctttgaa ctgagtcagt ggagctctactgaatactct acatggactg agagccgctg 2100 gaaagatatc cgtgcccgga tatttctcatcgccagcaaa gagcttgagt tgatcaccct 2160 gacagtgggc ttcggcatcc tcatcttctccctcatcgtc acctactgca tcaatgccaa 2220 agctgatgtc cttttcattg ctccccgggagccaggagct gtgtcatact gagsaggacc 2280 scagcttttc ttgccagctc agcagttcacttcctagagc atctgtccca ctgggacaca 2340 accactaatt tgtcactgga acctccctgggcctgtctca gattgggatt aacataaaag 2400 agtggaacta tccaaaagag acagggagaaataaataaat tgcctccctt cctccgctcc 2460 cctttcccat caccccttcc ccatttcctcttccttctct actcatgcca gattttggga 2520 ttacaaatag aagcttcttg ctcctgtttaactccctagt tacccaccct aatttgccct 2580 tcaggaccct tctacttttt ccttcctgccctgtacctct ctctgctcct cacccccacc 2640 cctgtaccca gccaccttcc tgactgggaaggacataaaa ggtttaatgt cagggtcaaa 2700 ctacattgag cccctgagga caggggcatctctgggctga gcctactgtc tccttcccac 2760 tgtcctttct ccaggccctc agatggcacattagggtggg cgtgctgcgg gtgggtatcc 2820 cacctccagc ccacagtgct cagttgtactttttattaag ctgtaatatc tatttttgtt 2880 tttgtctttt tcctttattc tttttgtaaatatatatata atgagtttca ttaaaataga 2940 ttatcccac 2949 <210> SEQ ID NO 14<211> LENGTH: 709 <212> TYPE: PRT <213> ORGANISM: human <400> SEQUENCE:14 Met Ala Thr Ala Gly Gly Gly Ser Gly Ala Asp Pro Gly Ser Arg Gly 1 510 15 Leu Leu Arg Leu Leu Ser Phe Cys Val Leu Leu Ala Gly Leu Cys Arg 2025 30 Gly Asn Ser Val Glu Arg Lys Ile Tyr Ile Pro Leu Asn Lys Thr Ala 3540 45 Pro Cys Val Arg Leu Leu Asn Ala Thr His Gln Ile Gly Cys Gln Ser 5055 60 Ser Ile Ser Gly Asp Thr Gly Val Ile His Val Val Glu Lys Glu Glu 6570 75 80 Asp Leu Gln Trp Val Leu Thr Asp Gly Pro Asn Pro Pro Tyr Met Val85 90 95 Leu Leu Glu Ser Lys His Phe Thr Arg Asp Leu Met Glu Lys Leu Lys100 105 110 Gly Arg Thr Ser Arg Ile Ala Gly Leu Ala Val Ser Leu Thr LysPro 115 120 125 Ser Pro Ala Ser Gly Phe Ser Pro Ser Val Gln Cys Pro AsnAsp Gly 130 135 140 Phe Gly Val Tyr Ser Asn Ser Tyr Gly Pro Glu Phe AlaHis Cys Arg 145 150 155 160 Glu Ile Gln Trp Asn Ser Leu Gly Asn Gly LeuAla Tyr Glu Asp Phe 165 170 175 Ser Phe Pro Ile Phe Leu Leu Glu Asp GluAsn Glu Thr Lys Val Ile 180 185 190 Lys Gln Cys Tyr Gln Asp His Asn LeuSer Gln Asn Gly Ser Ala Pro 195 200 205 Thr Phe Pro Leu Cys Ala Met GlnLeu Phe Ser His Met His Ala Val 210 215 220 Ile Ser Thr Ala Thr Cys MetArg Arg Ser Ser Ile Gln Ser Thr Phe 225 230 235 240 Ser Ile Asn Pro GluIle Val Cys Asp Pro Leu Ser Asp Tyr Asn Val 245 250 255 Trp Ser Met LeuLys Pro Ile Asn Thr Thr Gly Thr Leu Lys Pro Asp 260 265 270 Asp Arg ValVal Val Ala Ala Thr Arg Leu Asp Ser Arg Ser Phe Phe 275 280 285 Trp AsnVal Ala Pro Gly Ala Glu Ser Ala Val Ala Ser Phe Val Thr 290 295 300 GlnLeu Ala Ala Ala Glu Ala Leu Gln Lys Ala Pro Asp Val Thr Thr 305 310 315320 Leu Pro Arg Asn Val Met Phe Val Phe Phe Gln Gly Glu Thr Phe Asp 325330 335 Tyr Ile Gly Ser Ser Arg Met Val Tyr Asp Met Glu Lys Gly Lys Phe340 345 350 Pro Val Gln Leu Glu Asn Val Asp Ser Phe Val Glu Leu Gly GlnVal 355 360 365 Ala Leu Arg Thr Ser Leu Glu Leu Trp Met His Thr Asp ProVal Ser 370 375 380 Gln Lys Asn Glu Ser Val Arg Asn Gln Val Glu Asp LeuLeu Ala Thr 385 390 395 400 Leu Glu Lys Ser Gly Ala Gly Val Pro Ala ValIle Leu Arg Arg Pro 405 410 415 Asn Gln Ser Gln Pro Leu Pro Pro Ser SerLeu Gln Arg Phe Leu Arg 420 425 430 Ala Arg Asn Ile Ser Gly Val Val LeuAla Asp His Ser Gly Ala Phe 435 440 445 His Asn Lys Tyr Tyr Gln Ser IleTyr Asp Thr Ala Glu Asn Ile Asn 450 455 460 Val Ser Tyr Pro Glu Trp LeuSer Pro Glu Glu Asp Leu Asn Phe Val 465 470 475 480 Thr Asp Thr Ala LysAla Leu Ala Asp Val Ala Thr Val Leu Gly Arg 485 490 495 Ala Leu Tyr GluLeu Ala Gly Gly Thr Asn Phe Ser Asp Thr Val Gln 500 505 510 Ala Asp ProGln Thr Val Thr Arg Leu Leu Tyr Gly Phe Leu Ile Lys 515 520 525 Ala AsnAsn Ser Trp Phe Gln Ser Ile Leu Arg Gln Asp Leu Arg Ser 530 535 540 TyrLeu Gly Asp Gly Pro Leu Gln His Tyr Ile Ala Val Ser Ser Pro 545 550 555560 Thr Asn Thr Thr Tyr Val Val Gln Tyr Ala Leu Ala Asn Leu Thr Gly 565570 575 Thr Val Val Asn Leu Thr Arg Glu Gln Cys Gln Asp Pro Ser Lys Val580 585 590 Pro Ser Glu Asn Lys Asp Leu Tyr Glu Tyr Ser Trp Val Gln GlyPro 595 600 605 Leu His Ser Asn Glu Thr Asp Arg Leu Pro Arg Cys Val ArgSer Thr 610 615 620 Ala Arg Leu Ala Arg Ala Leu Ser Pro Ala Phe Glu LeuSer Gln Trp 625 630 635 640 Ser Ser Thr Glu Tyr Ser Thr Trp Thr Glu SerArg Trp Lys Asp Ile 645 650 655 Arg Ala Arg Ile Phe Leu Ile Ala Ser LysGlu Leu Glu Leu Ile Thr 660 665 670 Leu Thr Val Gly Phe Gly Ile Leu IlePhe Ser Leu Ile Val Thr Tyr 675 680 685 Cys Ile Asn Ala Lys Ala Asp ValLeu Phe Ile Ala Pro Arg Glu Pro 690 695 700 Gly Ala Val Ser Tyr 705<210> SEQ ID NO 15 <211> LENGTH: 2250 <212> TYPE: DNA <213> ORGANISM:mouse <400> SEQUENCE: 15 cccagcggag aggcaacatg gctacgacta ggggcggctctgggcctgac ccaggaagtc 60 ggggtcttct tcttctgtct ttttccgtgg tactggcaggattgtgtggg ggaaactcag 120 tggagaggaa aatctacatt cccttaaata aaacagctccttgtgtccgc ctgctcaacg 180 ccactcatca gattggctgc cagtcttcaa ttagtggggatacaggggtt atccacgtag 240 tggagaaaga agaagacctg aagtgggtgt tgaccgatggccccaacccc ccttacatgg 300 ttctgctgga gggcaagctc ttcaccagag atgtaatggagaagctgaag ggaacaacca 360 gtagaatcgc tggtcttgcc gtgactctag ccaagcccaactcaacttca agcttctctc 420 ctagtgtgca gtgcccaaat gatgggtttg gtatttactccaactcctac gggccagagt 480 ttgctcactg caagaaaaca ctgtggaatg aactgggcaacggcttggct tatgaagact 540 ttagtttccc catctttctt cttgaagatg agaacgaaaccaaggtcatc aagcagtgct 600 atcaagatca caacctgggt cagaatggct ctgcaccaagcttcccattg tgtgctatgc 660 agctcttctc acacatgcac gccgtcatca gcactgccacctgcatgcgg cgcagcttca 720 tccagagcac cttcagcatc aacccagaaa tcgtctgtgaccccttatct gactacaacg 780 tatggagcat gcttaagcct ataaatacat ctgtgggactagaacctgac gtcagggttg 840 tggttgcggc cacacggctg gatagccggt cctttttctggaatgtggcc ccaggggctg 900 aaagtgctgt agcctccttt gtcactcagc tggctgcagctgaagctttg cacaaggcac 960 ctgatgtgac cactctatcc cgaaatgtga tgtttgtcttcttccagggg gaaacttttg 1020 actacattgg cagctcacgg atggtctatg atatggagaacggcaagttt cccgtgcggc 1080 tcgagaacat cgactccttc gtggagctgg gacaggtggccctaagaact tcactagatc 1140 tctggatgca cacagatccc atgtctcaga aaaatgagtctgtgaaaaac caggtggagg 1200 atcttctggc cactctggag aagagcggtg ctggtgtccctgaagttgtc ctgaggagac 1260 tggcccagtc ccaggccctt ccaccttcat ctctacaacgatttcttcgg gctcgaaaca 1320 tctctggcgt ggtcctggct gaccactctg gctccttccacaatcggtat taccagagca 1380 tttatgacac ggctgagaac attaatgtga cctatcctgagtggcagagc ccagaagagg 1440 acctcaactt tgtgacagac actgccaagg cactggcgaatgtggccaca gtgctggcgc 1500 gtgcactgta tgagcttgca ggaggaacca acttcagcagctccatccag gctgatcccc 1560 agacagttac acgtctgctc tatgggttcc tggttaaagctaacaactca tggtttcagt 1620 cgatcctgaa acatgaccta aggtcctatt tggatgacaggcctcttcaa cactacatcg 1680 ccgtctccag ccctaccaac acgacttacg ttgtgcagtacgccttggca aacctgactg 1740 gcaaggcgac caacctcacc cgagagcagt gccaggatccaagtaaagtc ccaaatgaga 1800 gcaaggattt atatgaatac tcgtgggtac aaggcccttggaattccaac aggacagaga 1860 ggctcccaca gtgtgtgcgc tccacagtgc gactggccagggccttgtcc cctgcctttg 1920 aactgagtca gtggagctcc acagaatact ctacgtgggcggagagccgc tggaaagaca 1980 tccaagctcg gatattccta attgccagca aaaagcttgagttcatcacg ctgatcgtgg 2040 gcttcagcat ccttatcttc tctctcatcg tcacctactgcatcaatgcc aaagccgacg 2100 tccttttygt tgctccccga gagccaggag ctgtgtcttactgaagagga ctctagctct 2160 ccctgcctgc tctgaacttt acttcccaga ccaggtgtccggctgggaac aaaccactaa 2220 tttgtcactg gactgtctct gggcctgctt 2250 <210>SEQ ID NO 16 <211> LENGTH: 708 <212> TYPE: PRT <213> ORGANISM: mouse<400> SEQUENCE: 16 Met Ala Thr Thr Arg Gly Gly Ser Gly Pro Asp Pro GlySer Arg Gly 1 5 10 15 Leu Leu Leu Leu Ser Phe Ser Val Val Leu Ala GlyLeu Cys Gly Gly 20 25 30 Asn Ser Val Glu Arg Lys Ile Tyr Ile Pro Leu AsnLys Thr Ala Pro 35 40 45 Cys Val Arg Leu Leu Asn Ala Thr His Gln Ile GlyCys Gln Ser Ser 50 55 60 Ile Ser Gly Asp Thr Gly Val Ile His Val Val GluLys Glu Glu Asp 65 70 75 80 Leu Lys Trp Val Leu Thr Asp Gly Pro Asn ProPro Tyr Met Val Leu 85 90 95 Leu Glu Gly Lys Leu Phe Thr Arg Asp Val MetGlu Lys Leu Lys Gly 100 105 110 Thr Thr Ser Arg Ile Ala Gly Leu Ala ValThr Leu Ala Lys Pro Asn 115 120 125 Ser Thr Ser Ser Phe Ser Pro Ser ValGln Cys Pro Asn Asp Gly Phe 130 135 140 Gly Ile Tyr Ser Asn Ser Tyr GlyPro Glu Phe Ala His Cys Lys Lys 145 150 155 160 Thr Leu Trp Asn Glu LeuGly Asn Gly Leu Ala Tyr Glu Asp Phe Ser 165 170 175 Phe Pro Ile Phe LeuLeu Glu Asp Glu Asn Glu Thr Lys Val Ile Lys 180 185 190 Gln Cys Tyr GlnAsp His Asn Leu Gly Gln Asn Gly Ser Ala Pro Ser 195 200 205 Phe Pro LeuCys Ala Met Gln Leu Phe Ser His Met His Ala Val Ile 210 215 220 Ser ThrAla Thr Cys Met Arg Arg Ser Phe Ile Gln Ser Thr Phe Ser 225 230 235 240Ile Asn Pro Glu Ile Val Cys Asp Pro Leu Ser Asp Tyr Asn Val Trp 245 250255 Ser Met Leu Lys Pro Ile Asn Thr Ser Val Gly Leu Glu Pro Asp Val 260265 270 Arg Val Val Val Ala Ala Thr Arg Leu Asp Ser Arg Ser Phe Phe Trp275 280 285 Asn Val Ala Pro Gly Ala Glu Ser Ala Val Ala Ser Phe Val ThrGln 290 295 300 Leu Ala Ala Ala Glu Ala Leu His Lys Ala Pro Asp Val ThrThr Leu 305 310 315 320 Ser Arg Asn Val Met Phe Val Phe Phe Gln Gly GluThr Phe Asp Tyr 325 330 335 Ile Gly Ser Ser Arg Met Val Tyr Asp Met GluAsn Gly Lys Phe Pro 340 345 350 Val Arg Leu Glu Asn Ile Asp Ser Phe ValGlu Leu Gly Gln Val Ala 355 360 365 Leu Arg Thr Ser Leu Asp Leu Trp MetHis Thr Asp Pro Met Ser Gln 370 375 380 Lys Asn Glu Ser Val Lys Asn GlnVal Glu Asp Leu Leu Ala Thr Leu 385 390 395 400 Glu Lys Ser Gly Ala GlyVal Pro Glu Val Val Leu Arg Arg Leu Ala 405 410 415 Gln Ser Gln Ala LeuPro Pro Ser Ser Leu Gln Arg Phe Leu Arg Ala 420 425 430 Arg Asn Ile SerGly Val Val Leu Ala Asp His Ser Gly Ser Phe His 435 440 445 Asn Arg TyrTyr Gln Ser Ile Tyr Asp Thr Ala Glu Asn Ile Asn Val 450 455 460 Thr TyrPro Glu Trp Gln Ser Pro Glu Glu Asp Leu Asn Phe Val Thr 465 470 475 480Asp Thr Ala Lys Ala Leu Ala Asn Val Ala Thr Val Leu Ala Arg Ala 485 490495 Leu Tyr Glu Leu Ala Gly Gly Thr Asn Phe Ser Ser Ser Ile Gln Ala 500505 510 Asp Pro Gln Thr Val Thr Arg Leu Leu Tyr Gly Phe Leu Val Lys Ala515 520 525 Asn Asn Ser Trp Phe Gln Ser Ile Leu Lys His Asp Leu Arg SerTyr 530 535 540 Leu Asp Asp Arg Pro Leu Gln His Tyr Ile Ala Val Ser SerPro Thr 545 550 555 560 Asn Thr Thr Tyr Val Val Gln Tyr Ala Leu Ala AsnLeu Thr Gly Lys 565 570 575 Ala Thr Asn Leu Thr Arg Glu Gln Cys Gln AspPro Ser Lys Val Pro 580 585 590 Asn Glu Ser Lys Asp Leu Tyr Glu Tyr SerTrp Val Gln Gly Pro Trp 595 600 605 Asn Ser Asn Arg Thr Glu Arg Leu ProGln Cys Val Arg Ser Thr Val 610 615 620 Arg Leu Ala Arg Ala Leu Ser ProAla Phe Glu Leu Ser Gln Trp Ser 625 630 635 640 Ser Thr Glu Tyr Ser ThrTrp Ala Glu Ser Arg Trp Lys Asp Ile Gln 645 650 655 Ala Arg Ile Phe LeuIle Ala Ser Lys Lys Leu Glu Phe Ile Thr Leu 660 665 670 Ile Val Gly PheSer Ile Leu Ile Phe Ser Leu Ile Val Thr Tyr Cys 675 680 685 Ile Asn AlaLys Ala Asp Val Leu Phe Val Ala Pro Arg Glu Pro Gly 690 695 700 Ala ValSer Tyr 705 <210> SEQ ID NO 17 <211> LENGTH: 2942 <212> TYPE: DNA <213>ORGANISM: D. melanogaster <400> SEQUENCE: 17 tctgatatca tcgccactgtgctgggaatt cggcacgagc gcaacactgc aatttctgga 60 cgcgatttcg tgggaatcttcgatggaaat gcgtctgaat gcggcttcca tatggctgtt 120 aatactgtcg tatggagcaactattgctca aggagaaaga acccgcgata agatgtacga 180 gcccattgga ggagctagctgtttccgacg gctgaatggc acccatcaga caggctgttc 240 ctcaacctac tccggttccgtgggcgtact acatctaata aacgtcgagg ccgacctgga 300 atttcttctt agcagcccaccatctccacc ttacgccccc atgataccac ctcacctgtt 360 cacacgtaac aacctgatgcgcctaaagga agccggacca aagaacattt ctgtggtgct 420 gctgatcaac cgcacgaaccagatgaagca gttctcgcac gaactcaact gccccaatca 480 gtacagcggc ctgaacagcaccagtgagac ctgcgacgcc agcaatccag ccaaaaactg 540 gaatccctgg ggcactggacttctgcacga ggactttccc tttcctatct attacatagc 600 cgatttggat caggtcaccaagctagagaa gtgctttcag gactttaaca accataacta 660 cgagacgcac gcgctgcgtagcttgtgcgc cgtcgaggtc aagtccttta tgtccgccgc 720 tgtcaacacc gaggtctgtatgcgccgcac caacttcatc aataatcttg gaggaagcaa 780 gtactgcgat ccgctcgagggacggaatgt ttcgccacct tgtacccccg aaagccagca 840 atcggaaaca actttggagacagtccatac gaatgaaaag ttcatattag taacctgtcg 900 cctggacacc accaccatgttcgatggcgt cggtcttgga gccatggact ccctcatggg 960 atttgctgtt ttcactcatgtggcgtatct attaaaacaa ctacttccgc cgcaaagcaa 1020 agaccttcat aatgtcctctttgtgacttt taatggcgaa tcctatgact acattggttc 1080 tcaaagattt gtatacgacatggagaaact tcaatttcct actgaatcca caggcacgcc 1140 tccgattgcc tttgacaatattgacttcat gctggacatc gggacactgg atgacatatc 1200 gaatattaag ctgcatgcgttaaatggaac gactttggct cagcaaattc tagagcggct 1260 aaataactat gcgaagtcgccacgctatgg ctttaacctg aacattcagt ccgagatgag 1320 cgctcactta ccacctacgtcggcgcaatc atttctgcga cgtgatccaa acttcaatgc 1380 attgattcta aacgctcgtccaacgaacaa gtattatcat tccacatacg atgacgcgga 1440 taacgtggac ttcacctatgcgaacacaag caaggatttc acccagctga cggaagttaa 1500 tgactttaaa agcttgaacccagattcact gcaaatgaaa gtgcgcaacg tttcctctat 1560 tgtggccatg gccctatatcagacaataac tggaaaggag tacactggca ccaaggtggc 1620 caaccctctg atggcagatgagttccttta ctgtttcctg caatcggcgg actgcccact 1680 ctttaaggcc gcatcttatccgggcagtca gctcaccaat ttgcctccga tgcgctacat 1740 aagcgtcttg ggtggctctcaagagtcgtc gggctatacg tatagattgc tgggctatct 1800 cttgtcacaa ctgcagccagacattcacag agataactgc accgacttgc cgctgcacta 1860 tttcgccgga ttcaacaatatcggagagtg tcgcctcacc acgcagaact acagtcacgc 1920 cctgagtcca gcttttcttattgatggcta cgattggagt tccggcatgt attccacttg 1980 gactgaatcc acctggtcacagttcagtgc acgcatcttc ctgcgcccgt ccaatgtgca 2040 ccaggtcaca actctaagcgttggcatagt ggtgctgata atatccttct gtttggtgta 2100 tataatcagc tcacgatcggaagtcctctt tgaggatttg ccggcaagca atgccgcatt 2160 atttggttga tgtcacaactgccacagcga cgaaatcatc gcgttcagca gctcattcca 2220 tagatttctg catgcgtaaactaaacgtta cttgtaaacc aatcgattaa gaatttctga 2280 ttgtgccctt ttagatcgccgcggccgaca gcctgttaaa tcttcaaaga atatctgatc 2340 acgtgccgaa gatgattggttgctgaatat gatctaaaac aaaaaacaga cttaacaaag 2400 acactaaaat gatattctaactcgtcttat ttaaaacatt aagcaaacgt ttatttatat 2460 gtatttttgt attttaaagtagtaaattag cttctatcaa ctacgttgta tcatatatag 2520 acatttaacc aattgcgacaaaattctttc cacttgtccg gccctttttg cattgtacat 2580 aggattaacc aacccaattgaacctctgat aatgccaagg aagagatgtc tgtacaaata 2640 ttgacaagaa actgctataacttataaatc actggaaata tttatacctt ctgcatctat 2700 tgcgatactg aacttaatgatctgaaatca ttacttcata gaagacaaat aattattata 2760 acgactttaa attatatatgtttaataaat tttgataagg tgtaaagcaa tgtcctgtta 2820 tctagttagg ttattttcaaggcaattatt cacagctctc agattccaac gattgatgta 2880 gtttaatctc aactctttaccaaagaagtc catttgtact agtgtaaaag atattttcaa 2940 ta 2942 <210> SEQ ID NO18 <211> LENGTH: 695 <212> TYPE: PRT <213> ORGANISM: D. melanogaster<400> SEQUENCE: 18 Met Glu Met Arg Leu Asn Ala Ala Ser Ile Trp Leu LeuIle Leu Ser 1 5 10 15 Tyr Gly Ala Thr Ile Ala Gln Gly Glu Arg Thr ArgAsp Lys Met Tyr 20 25 30 Glu Pro Ile Gly Gly Ala Ser Cys Phe Arg Arg LeuAsn Gly Thr His 35 40 45 Gln Thr Gly Cys Ser Ser Thr Tyr Ser Gly Ser ValGly Val Leu His 50 55 60 Leu Ile Asn Val Glu Ala Asp Leu Glu Phe Leu LeuSer Ser Pro Pro 65 70 75 80 Ser Pro Pro Tyr Ala Pro Met Ile Pro Pro HisLeu Phe Thr Arg Asn 85 90 95 Asn Leu Met Arg Leu Lys Glu Ala Gly Pro LysAsn Ile Ser Val Val 100 105 110 Leu Leu Ile Asn Arg Thr Asn Gln Met LysGln Phe Ser His Glu Leu 115 120 125 Asn Cys Pro Asn Gln Tyr Ser Gly LeuAsn Ser Thr Ser Glu Thr Cys 130 135 140 Asp Ala Ser Asn Pro Ala Lys AsnTrp Asn Pro Trp Gly Thr Gly Leu 145 150 155 160 Leu His Glu Asp Phe ProPhe Pro Ile Tyr Tyr Ile Ala Asp Leu Asp 165 170 175 Gln Val Thr Lys LeuGlu Lys Cys Phe Gln Asp Phe Asn Asn His Asn 180 185 190 Tyr Glu Thr HisAla Leu Arg Ser Leu Cys Ala Val Glu Val Lys Ser 195 200 205 Phe Met SerAla Ala Val Asn Thr Glu Val Cys Met Arg Arg Thr Asn 210 215 220 Phe IleAsn Asn Leu Gly Gly Ser Lys Tyr Cys Asp Pro Leu Glu Gly 225 230 235 240Arg Asn Val Ser Pro Pro Cys Thr Pro Glu Ser Gln Gln Ser Glu Thr 245 250255 Thr Leu Glu Thr Val His Thr Asn Glu Lys Phe Ile Leu Val Thr Cys 260265 270 Arg Leu Asp Thr Thr Thr Met Phe Asp Gly Val Gly Leu Gly Ala Met275 280 285 Asp Ser Leu Met Gly Phe Ala Val Phe Thr His Val Ala Tyr LeuLeu 290 295 300 Lys Gln Leu Leu Pro Pro Gln Ser Lys Asp Leu His Asn ValLeu Phe 305 310 315 320 Val Thr Phe Asn Gly Glu Ser Tyr Asp Tyr Ile GlySer Gln Arg Phe 325 330 335 Val Tyr Asp Met Glu Lys Leu Gln Phe Pro ThrGlu Ser Thr Gly Thr 340 345 350 Pro Pro Ile Ala Phe Asp Asn Ile Asp PheMet Leu Asp Ile Gly Thr 355 360 365 Leu Asp Asp Ile Ser Asn Ile Lys LeuHis Ala Leu Asn Gly Thr Thr 370 375 380 Leu Ala Gln Gln Ile Leu Glu ArgLeu Asn Asn Tyr Ala Lys Ser Pro 385 390 395 400 Arg Tyr Gly Phe Asn LeuAsn Ile Gln Ser Glu Met Ser Ala His Leu 405 410 415 Pro Pro Thr Ser AlaGln Ser Phe Leu Arg Arg Asp Pro Asn Phe Asn 420 425 430 Ala Leu Ile LeuAsn Ala Arg Pro Thr Asn Lys Tyr Tyr His Ser Thr 435 440 445 Tyr Asp AspAla Asp Asn Val Asp Phe Thr Tyr Ala Asn Thr Ser Lys 450 455 460 Asp PheThr Gln Leu Thr Glu Val Asn Asp Phe Lys Ser Leu Asn Pro 465 470 475 480Asp Ser Leu Gln Met Lys Val Arg Asn Val Ser Ser Ile Val Ala Met 485 490495 Ala Leu Tyr Gln Thr Ile Thr Gly Lys Glu Tyr Thr Gly Thr Lys Val 500505 510 Ala Asn Pro Leu Met Ala Asp Glu Phe Leu Tyr Cys Phe Leu Gln Ser515 520 525 Ala Asp Cys Pro Leu Phe Lys Ala Ala Ser Tyr Pro Gly Ser GlnLeu 530 535 540 Thr Asn Leu Pro Pro Met Arg Tyr Ile Ser Val Leu Gly GlySer Gln 545 550 555 560 Glu Ser Ser Gly Tyr Thr Tyr Arg Leu Leu Gly TyrLeu Leu Ser Gln 565 570 575 Leu Gln Pro Asp Ile His Arg Asp Asn Cys ThrAsp Leu Pro Leu His 580 585 590 Tyr Phe Ala Gly Phe Asn Asn Ile Gly GluCys Arg Leu Thr Thr Gln 595 600 605 Asn Tyr Ser His Ala Leu Ser Pro AlaPhe Leu Ile Asp Gly Tyr Asp 610 615 620 Trp Ser Ser Gly Met Tyr Ser ThrTrp Thr Glu Ser Thr Trp Ser Gln 625 630 635 640 Phe Ser Ala Arg Ile PheLeu Arg Pro Ser Asn Val His Gln Val Thr 645 650 655 Thr Leu Ser Val GlyIle Val Val Leu Ile Ile Ser Phe Cys Leu Val 660 665 670 Tyr Ile Ile SerSer Arg Ser Glu Val Leu Phe Glu Asp Leu Pro Ala 675 680 685 Ser Asn AlaAla Leu Phe Gly 690 695 <210> SEQ ID NO 19 <211> LENGTH: 17 <212> TYPE:PRT <213> ORGANISM: human <400> SEQUENCE: 19 Ala Arg Leu Ala Arg Ala LeuSer Pro Ala Phe Glu Leu Ser Gln Trp 1 5 10 15 Ser

We claim:
 1. An isolated nucleic acid which encodes humanpresenilin-associated membrane protein (PAMP) as set forth in SEQ IDNO:14.
 2. The isolated nucleic acid of claim 1 which comprises anucleotide sequence encoding human PAMP as set forth in SEQ ID NO:13. 3.A vector comprising the nucleic acid of claim 1 operatively associatedwith an expression control sequence.
 4. An isolated cell comprising thevector of claim
 3. 5. A method for producing human PAMP, which methodcomprises culturing the cell of claim 4 under conditions treat permitexpression of the human PAMP.
 6. An isolated nucleic acid encoding amutant PAMP, wherein the mutant PAMP has a mutation in an amino acidresidue corresponding to an amino acid selected from the groupconsisting of C230, D336, Y337, and both D336 and Y337, of human PAMP asset forth in SEQ ID NO:14.
 7. The isolated nucleic acid of claim 6,wherein the mutation is selected from the group consisting of C230A,D336A, Y337A, and both D336A and Y337A.
 8. A vector comprising thenucleic acid of claim 6, operatively associated with an expressioncontrol sequence.
 9. An isolated call transfected with the vector ofclaim
 8. 10. A method for producing a mutant of SEQ ID NO:14, whichmethod comprises culturing the cell of claim 9 under conditions thatpermit expression of the mutant PAMP.
 11. An isolated nucleic acidencoding a mutant human PAMP less capable than human PAMP as set forthin SEQ ID NO:14 of interacting with a presenilin protein, wherein themutant PAMP has a deletion of an amino acid sequence corresponding toΔ312-369 of Δ312-340 of SEQ ID NO:14.
 12. A vector comprising thenucleic acid of claim 11, operatively associated with an expressioncontrol sequence.
 13. An isolated cell transfected with the vector ofclaim
 12. 14. A method for producing a mutant of SEQ ID NO:14, whichmethod comprises culturing the cell of claim 13 under conditions thatpermit expression of the mutant PAMP.